Nucleic acid molecules encoding activin receptor type IIA variants

ABSTRACT

The invention features polypeptides that include an extracellular ActRIIa variant. In some embodiments, a polypeptide of the invention includes an extracellular ActRIIa variant fused to an Fc domain monomer or moiety. The invention also features pharmaceutical compositions and methods of using the polypeptides to treat diseases and conditions involving weakness and atrophy of muscles, e.g., Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, inclusion body myositis, amyotrophic lateral sclerosis, sarcopenia; or cancer cachexia; or metabolic diseases, e.g., obesity, Type-1 diabetes, or Type-2 diabetes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created May 21, 2021, isnamed 51184-002005_Sequence_Listing_052121_ST25 and is 133,894 bytes insize.

BACKGROUND OF THE INVENTION

Duchenne muscular dystrophy (DMD), facioscapulohumeral musculardystrophy (FSHD), inclusion body myositis (IBM), and amyotrophic lateralsclerosis (ALS) are examples of muscle diseases that involve weaknessand atrophy of muscles and/or motor neurons that control voluntarymuscle movements. DMD is caused by mutations in the X-linked dystrophingene and characterized by progressive muscle degeneration and weaknessin all skeletal muscles. FSHD particularly affects skeletal muscles ofthe face, shoulders, upper arms, and lower legs. IBM is an inflammatorymuscle disease that mainly affects muscles of the thighs and muscles ofthe arms that control finger and wrist flexion. ALS is a motor neurondisease characterized by stiff muscles, muscle twitching, and muscleatrophy throughout the body due to the degeneration of the motorneurons. Efforts to improve treatment and survival of subjects havingthese devastating muscle diseases have not been successful.

Excess body weight is an increasing problem in the United States, with aprevalence of approximately 25% of the population. Increased visceraland subcutaneous fact causes dysfunction of various organs. Excessivebody weight is a risk factor for an array of complications, includingobesity, diabetes (e.g., Type-1 and Type-2 diabetes), cardiovasculardisease, and several forms of cancer. Insulin resistance is alsoassociated with obesity and occurs when pancreatic tissues require anelevated amount of insulin. Once pancreatic p cells can no longerproduce sufficient insulin to meet the demand, hyperglycemia occurs andType-2 diabetes develops. Adipocytes, which are increased in obesity,are believed to play a role in this process. Despite the prevalence ofobesity and metabolic diseases such as diabetes (e.g., Type-1 and Type-2diabetes) and insulin resistance, few therapeutic options are available.

There exists a need for novel treatments for these muscular andmetabolic diseases.

SUMMARY OF THE INVENTION

The present invention features polypeptides that include anextracellular activin receptor type IIa (ActRIIa) variant. In someembodiments, a polypeptide of the invention includes an extracellularActRIIa variant fused to the N- or C-terminus of an Fc domain monomer ormoiety. Such moieties may be attached by amino acid or other covalentbonds and may increase stability of the polypeptide. A polypeptideincluding an extracellular ActRIIa variant fused to an Fc domain monomermay also form a dimer (e.g., a homodimer or heterodimer) through theinteraction between two Fc domain monomers. The polypeptides of theinvention may be used to increase muscle mass and strength in a subjecthaving a disease or condition involving weakness and atrophy of muscles,e.g., Duchenne muscular dystrophy (DMD), facioscapulohumeral musculardystrophy (FSHD), inclusion body myositis (IBM), amyotrophic lateralsclerosis (ALS), sarcopenia, or cancer cachexia. The polypeptides of theinvention may also be used to reduce body weight, reduce body fat,increase glucose clearance, increase insulin sensitivity, or reducefasting insulin levels in a subject having or at risk of developing ametabolic disease, e.g., obesity, Type-1 diabetes, or Type-2 diabetes.Further, the polypeptides of the invention may also be used to affectmyostatin, activin, and/or bone morphogenetic protein 9 (BMP9) signalingin a subject having a risk of developing or having a disease orcondition involving weakness and atrophy of muscles or a metabolicdisease.

In one aspect, the invention features a polypeptide including anextracellular activin receptor type IIa (ActRIIa) variant, the varianthaving a sequence ofGAILGRSETQECLX₁X₂NANWX₃X₄X₅X₆TNQTGVEX₇CX₈GXX₁₀X₁₁X₁₂X₁₃X₁₄HCX₁₅ATWX₁₆NISGSIEIVX₁₇X₁₈GCX₁₉X₂₀X₂₁DX₂₂NCYDRTDCVEX₂₃X₂₄X₂₅X₂₆PX₂₇VYFCCCEGNMCNEKFSYFPEMEVTQPTS(SEQ ID NO: 1), wherein X₁ is F or Y; X₂ is F or Y; X₃ is E or A; X₄ isK or L; X₅ is D or E; X₆ is R or A; X₇ is P or R; X₈ is Y or E; X₉ is Dor E; X₁₀ is K or Q; X₁ is D or A; X₁₂ is K or A; X₁₃ is R or A; X₁₄ isR or L; X₁₅ is F or Y; X₁₆ is K, R, or A; X₁₇ is K, A, Y, F, or I; X₁₈is Q or K; X₁₉ is W or A; X₂₀ is L or A; X₂₁ is D, K, R, A, F, G, M, N,or I; X₂₂ is I, F, or A; X₂₃ is K or T; X₂₄ is K or E; X₂₅ is D or E;X₂₆ is S or N; and X₂₇ is E or Q, and wherein the variant has at leastone amino acid substitution relative to a wild-type extracellularActRIIa having the sequence of SEQ ID NO: 73 or an extracellular ActRIIahaving any one of the sequences of SEQ ID NOs: 76-96.

In some embodiments, the variant has a sequence ofGAILGRSETQECLFX₂NANWX₃X₄X₅X₆TNQTGVEXCXGXKX₁₁X₁₂X₁₃X₁₄HCX₁₅ATWX₁₆NISGSIEIVX₁₇X₁₈GCX₁₉X₂₀X₂₁DX₂₂NCYDRTDCVEX₂₃X₂₄X₂₅X₂PX₂₇VYFCCCEGNMCNEKFSYFPEMEVTQPTS(SEQ ID NO: 2), wherein X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₉, X₁₁, X₁₂, X₁₃,X₁₄, X₁₅, X₁₆, X₁₇, X₁₈, X₁₉, X₂₀, X₂₁, X₂₂, X₂₃, X₂₄, X₂₅, X₂₆, and X₂₇are defined as above.

In some embodiments, the variant has a sequence of

(SEQ ID NO: 3) GAILGRSETQECLFX₂NANWEX₄X₅RTNQTGVEX₇CX₈GX₉KDKRX₁₄HCX₁₅ATWX₁₆NISGSIEIVKX₁₈GCWLDDX₂₂NCYDRTDCVEX₂₃X₂₄X₂₅X₂₆PX₂₇VYFCCCEGNMCNEKFSYFPEMEVTQPTS,wherein X₂, X₄, X₇, X₈, X₉, X₁₄, X₁₅, X₁₆, X₁₈, X₂₂, X₂₃, X₂₄, X₂₅, X₂₆,and X₂₇ are defined as above.

In some embodiments, the variant has a sequence of

(SEQ ID NO: 4) GAILGRSETQECLFX₂NANWEX₄DRTNQTGVEX₇CX₈GX₉KDKRX₁₄HCX₁₅ATWX¹⁶NISGSIEIVKX₁₈GCWLDDX₂₂NCYDRTDCVEX₂₃KX₂₅X₂₆PX₂₇VYFCCCEGNMCNEKFSYFPEMEVTQPTS,wherein X₂, X₄, X₇, X₈, X₉, X₁₄, X₁₅, X₁₆, X₁₈, X₂₂, X₂₃, X₂₅, X₂₆, andX₂₇ are defined as above.

In some embodiments, the variant has a sequence ofGAILGRSETQECLFX₂NANWEX₄DRTNQTGVEPCX₈GX₉KDKRX₁₄HCFATWKNISGSIEIVKX₁₈GCWLDDINCYDRTDCVEX₂₃KX₂₅X₂₆PX₂₇VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 5),wherein X₂, X₄, X₈, X₉, X₁₄, X₁₈, X₂₃, X₂₅, X₂₆, and X₂₇ are defined asabove.

In any of the aforementioned embodiments, X₁ is F or Y. In any of theaforementioned embodiments, X₂ is F or Y. In any of the aforementionedembodiments, X₃ is E or A. In any of the aforementioned embodiments, X₄is K or L. In any of the aforementioned embodiments, X_(s) is D or E. Inany of the aforementioned embodiments, X₆ is R or A. In any of theaforementioned embodiments, X₇ is P or R. In any of the aforementionedembodiments, X₈ is Y or E. In any of the aforementioned embodiments, X₉is D or E. In any of the aforementioned embodiments, X₁₀ is K or Q. Inany of the aforementioned embodiments, X₁₁ is D or A. In any of theaforementioned embodiments, X₁₂ is K or A. In any of the aforementionedembodiments, X₁₃ is R or A. In any of the aforementioned embodiments,X₁₄ is R or L. In any of the aforementioned embodiments, X₁₅ is F or Y.In any of the aforementioned embodiments, X₁₆ is K, R, or A. In any ofthe aforementioned embodiments, X₁₇ is K, A, Y, F, or I. In any of theaforementioned embodiments, X₁₈ is Q or K. In any of the aforementionedembodiments, X_(1e) is W or A. In any of the aforementioned embodiments,X₂₀ is L or A. In any of the aforementioned embodiments, X₂₁ is D, K, R,A, F, G, M, N, or I. In any of the aforementioned embodiments, X₂₂ is I,F, or A. In any of the aforementioned embodiments, X₂₃ is K or T. In anyof the aforementioned embodiments, X₂₄ is K or E. In any of theaforementioned embodiments, X₂₅ is D or E. In any of the aforementionedembodiments, X₂₆ is S or N. In any of the aforementioned embodiments,X₂₇ is E or Q. In any of the aforementioned embodiments, X₂₃ is T, X₂₄is E, X₂₅ is E, and X₂₆ is N. In any of the aforementioned embodiments,X₂₃ is T, X₂₄ is K, X₂₅ is E, and X₂₆ is N. In any of the aforementionedembodiments, X₁₇ is K.

In any of the aforementioned embodiments, the variant has the sequenceof any one of SEQ ID NOs: 6-72.

In any of the aforementioned embodiments, the amino acid at position X₂₄may be replaced with the amino acid K.

In any of the aforementioned embodiments, the amino acid at position X₂₄may be replaced with the amino acid E.

In any of the aforementioned embodiments, a polypeptide described hereinmay further include a C-terminal extension of one or more amino acids(e.g., 1, 2, 3, 4, 5, 6, or more amino acids). In some embodiments, theC-terminal extension is amino acid sequence NP. In some embodiments, theC-terminal extension is amino acid sequence NPVTPK (SEQ ID NO: 155).

In any of the aforementioned embodiments, a polypeptide described hereinmay further include a moiety fused or covalently linked to theC-terminus of the polypeptide. In some embodiments, the moiety increasesstability or improves the pharmacokinetics of the polypeptide. In someembodiments, the moiety is an Fc domain, an albumin-binding peptide, afibronectin domain, or human serum albumin.

In any of the aforementioned embodiments, a polypeptide described hereinmay further include an Fc domain monomer fused to the C-terminus of thepolypeptide by way of a linker. In some embodiments, the polypeptidethat includes an extracellular ActRIIa variant described herein fused toan Fc domain monomer may form a dimer (e.g., a homodimer or heterodimer)through the interaction between two Fc domain monomers. In someembodiments, the Fc domain monomer has the sequence of SEQ ID NO: 97 Inany of the aforementioned embodiments, a polypeptide described hereinmay further include an Fc domain fused to the C-terminus of thepolypeptide by way of a linker. In some embodiments, the Fc domain is awild-type Fc domain. In some embodiments, the wild-type Fc domain hasthe sequence of SEQ ID NO: 151. In some embodiments, the Fc domaincontains one or more amino acid substitutions.

In some embodiments, the Fc domain containing one or more amino acidsubstitutions does not form a dimer.

In any of the aforementioned embodiments, a polypeptide described hereinmay further include an albumin-binding peptide fused to the C-terminusof the polypeptide by way of a linker. In some embodiments, thealbumin-binding peptide has the sequence of SEQ ID NO: 152.

In any of the aforementioned embodiments, a polypeptide described hereinmay further include a fibronectin domain fused to the C-terminus of thepolypeptide by way of a linker. In some embodiments, the fibronectindomain peptide has the sequence of SEQ ID NO: 153.

In any of the aforementioned embodiments, a polypeptide described hereinmay further include a human serum albumin fused to the C-terminus of thepolypeptide by way of a linker. In some embodiments, the human serumalbumin has the sequence of SEQ ID NO: 154.

In some embodiments, the linker is an amino acid spacer. In someembodiments, the amino acid spacer is GGG, GGGA (SEQ ID NO: 98), GGGG(SEQ ID NO: 100), GGGAG (SEQ ID NO: 130), GGGAGG (SEQ ID NO: 131), orGGGAGGG (SEQ ID NO: 132).

In some embodiments, the amino acid spacer is GGGS (SEQ ID NO: 99),GGGGA (SEQ ID NO: 101), GGGGS (SEQ ID NO: 102), GGGGG (SEQ ID NO: 103),GGAG (SEQ ID NO: 104), GGSG (SEQ ID NO: 105), AGGG (SEQ ID NO: 106),SGGG (SEQ ID NO: 107), GAGA (SEQ ID NO: 108), GSGS (SEQ ID NO: 109),GAGAGA (SEQ ID NO: 110), GSGSGS (SEQ ID NO: 111), GAGAGAGA (SEQ ID NO:112), GSGSGSGS (SEQ ID NO: 113), GAGAGAGAGA (SEQ ID NO: 114), GSGSGSGSGS(SEQ ID NO: 115), GAGAGAGAGAGA (SEQ ID NO: 116), and GSGSGSGSGSGS (SEQID NO: 117), GGAGGA (SEQ ID NO: 118), GGSGGS (SEQ ID NO: 119), GGAGGAGGA(SEQ ID NO: 120), GGSGGSGGS (SEQ ID NO: 121), GGAGGAGGAGGA (SEQ ID NO:122), GGSGGSGGSGGS (SEQ ID NO: 123), GGAGGGAG (SEQ ID NO: 124), GGSGGGSG(SEQ ID NO: 125), GGAGGGAGGGAG (SEQ ID NO: 126), and GGSGGGSGGGSG (SEQID NO: 127), GGGGAGGGGAGGGGA (SEQ ID NO: 128), GGGGSGGGGSGGGGS (SEQ IDNO: 129), AAAL (SEQ ID NO: 133), AAAK (SEQ ID NO: 134), AAAR (SEQ ID NO:135), EGKSSGSGSESKST (SEQ ID NO: 136), GSAGSAAGSGEF (SEQ ID NO: 137),AEAAAKEAAAKA (SEQ ID NO: 138), KESGSVSSEQLAQFRSLD (SEQ ID NO: 139),GENLYFQSGG (SEQ ID NO: 140), SACYCELS (SEQ ID NO: 141), RSIAT (SEQ IDNO: 142), RPACKIPNDLKQKVMNH (SEQ ID NO: 143),GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 144), AAANSSIDLISVPVDSR(SEQ ID NO: 145), GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 146),EAAAK (SEQ ID NO: 147), or PAPAP (SEQ ID NO: 148).

In any of the aforementioned embodiments, the polypeptide describedherein has a serum half-life of at least 7 days.

In any of the aforementioned embodiments, the polypeptide describedherein binds to human bone morphogenetic protein 9 (BMP9) with a K_(D)of 200 pM or higher. In some embodiments, the polypeptide binds toactivin and/or myostatin and has reduced (e.g., weak) binding to humanBMP9. In some embodiments, the polypeptide does not substantially bindto human BMP9.

In any of the aforementioned embodiments, the polypeptide describedherein binds to human activin A with a K_(D) of 800 pM or less.

In any of the aforementioned embodiments, the polypeptide describedherein binds to human activin B with a K_(D) of approximately 800 pM orless.

In any of the aforementioned embodiments, the polypeptide describedherein binds to human GDF-11 with a K_(D) of approximately 5 pM orhigher.

In another aspect, the invention features a nucleic acid moleculeencoding a polypeptide described herein (e.g., a polypeptide includingan extracellular ActRIIa variant having a sequence of any one of SEQ IDNOs: 1-72 (e.g., SEQ ID NOs: 6-72)). In another aspect, the inventionalso features a vector including the nucleic acid molecule describedherein.

In another aspect, the invention features a host cell that expresses apolypeptide described herein, wherein the host cell includes a nucleicacid molecule or a vector described in the previous two aspects, whereinthe nucleic acid molecule or vector is expressed in the host cell.

In another aspect, the invention features a method of preparing apolypeptide described herein, wherein the method includes: a) providinga host cell including a nucleic acid molecule or a vector describedherein, and b) expressing the nucleic acid molecule or vector in thehost cell under conditions that allow for the formation of thepolypeptide.

In another aspect, the invention features a pharmaceutical compositionincluding a polypeptide, nucleic acid molecule, or vector describedherein and one or more pharmaceutically acceptable carriers orexcipients. In some embodiments of the pharmaceutical composition, thepolypeptide, nucleic acid molecule, or vector is in a therapeuticallyeffective amount.

In another aspect, the invention also features a construct including twoidentical polypeptides (e.g., a homodimer) each including anextracellular ActRIIa variant having a sequence of any one of SEQ IDNOs: 1-72 (e.g., SEQ ID NOs: 6-72)) fused to the N- or C-terminus of anFc domain monomer (e.g., the sequence of SEQ ID NO: 97). The two Fcdomain monomers in the two polypeptides interact to form an Fc domain inthe construct.

In another aspect, the invention also features a construct including twodifferent polypeptides (e.g., a heterodimer) each including anextracellular ActRIIa variant having a sequence of any one of SEQ IDNOs: 1-72 (e.g., SEQ ID NOs: 6-72)) fused to the N- or C-terminus of anFc domain monomer (e.g., the sequence of SEQ ID NO: 97). The two Fcdomain monomers in the two polypeptides interact to form an Fc domain inthe construct.

In another aspect, the invention features a method of increasing musclemass in a subject in need thereof. The method includes administering tothe subject a therapeutically effective amount of a polypeptide, nucleicacid molecule, or vector described herein or a pharmaceuticalcomposition described herein.

In some embodiments of the method of increasing muscle mass in asubject, the subject has Duchenne muscular dystrophy (DMD),facioscapulohumeral muscular dystrophy (FSHD), inclusion body myositis(IBM), amyotrophic lateral sclerosis (ALS), sarcopenia, or cancercachexia.

In another aspect, the invention features a method of affectingmyostatin, activin, and/or BMP9 signaling (e.g., reducing or inhibitingthe binding of myostatin, activin, and/or BMP9 to their receptors) in asubject having a disease or condition involving weakness and atrophy ofmuscles, wherein method includes administering to the subject atherapeutically effective amount of a polypeptide, nucleic acidmolecule, or vector described herein or a pharmaceutical compositiondescribed herein. In some embodiments of this aspect, the disease orcondition is DMD, FSHD, IBM, ALS, sarcopenia, or cancer cachexia.

In another aspect, the invention features a method of treating a subjecthaving DMD by administering to the subject a therapeutically effectiveamount of a polypeptide, nucleic acid molecule, or vector describedherein or a pharmaceutical composition described herein.

In another aspect, the invention features a method of treating a subjecthaving FSHD by administering to the subject a therapeutically effectiveamount of a polypeptide, nucleic acid molecule, or vector describedherein or a pharmaceutical composition described herein.

In another aspect, the invention features a method of treating a subjecthaving IBM by administering to the subject a therapeutically effectiveamount of a polypeptide, nucleic acid molecule, or vector describedherein or a pharmaceutical composition described herein.

In another aspect, the invention features a method of treating a subjecthaving ALS by administering to the subject a therapeutically effectiveamount of a polypeptide, nucleic acid molecule, or vector describedherein or a pharmaceutical composition described herein.

In another aspect, the invention features a method of reducing body fatin a subject in need thereof by administering to the subject atherapeutically effective amount of a polypeptide, a nucleic acidmolecule, or vector described herein or a pharmaceutical compositiondescribed herein.

In another aspect, the invention features a method of reducing bodyweight in a subject in need thereof by administering to the subject atherapeutically effective amount of a polypeptide, a nucleic acidmolecule, or vector described herein or a pharmaceutical compositiondescribed herein.

In another aspect, the invention features a method of reducing bloodglucose in a subject in need thereof by administering to the subject atherapeutically effective amount of a polypeptide, a nucleic acidmolecule, or vector described herein or a pharmaceutical compositiondescribed herein.

In another aspect, the invention features a method of increasing insulinsensitivity in a subject in need thereof, by administering to thesubject a therapeutically effective amount of a polypeptide, a nucleicacid molecule, or vector described herein or a pharmaceuticalcomposition described herein.

In some embodiments of any of the above aspects, the subject has or isat risk of developing a metabolic disease. In some embodiments, themetabolic disease is selected from the group including obesity, Type-1diabetes, and Type-2 diabetes.

In another aspect, the invention features a method of affectingmyostatin, activin, and/or BMP9 signaling (e.g., reducing or inhibitingthe binding of myostatin, activin, and/or BMP9 to their receptors) in asubject having or at risk of developing a metabolic disease byadministering to the subject a therapeutically effective amount of apolypeptide, a nucleic acid molecule, or vector described herein or apharmaceutical composition described herein.

In another aspect, the invention features a method of treating and/orpreventing a metabolic disease in a subject by administering to thesubject a therapeutically effective amount of a polypeptide, a nucleicacid molecule, or vector described herein or a pharmaceuticalcomposition described herein.

In some embodiments of any of the above aspect, the metabolic disease isselected from the group including obesity, Type-1 diabetes, and Type-2diabetes. In some embodiments of any of the above aspects, the metabolicdisease is obesity. In some embodiments of any of the above aspects, themetabolic disease is Type-1 diabetes. In some embodiments of any of theabove aspects, the metabolic disease is Type-2 diabetes.

In some embodiments of any of the above aspects, the method reduces bodyweight and/or percentage of body weight gain of said subject. In someembodiments of any of the above aspects, the method reduces amount ofbody fat and/or percentage of body fat of said subject. In someembodiments of any of the above aspects, the method does not affect theappetite for food intake of said subject. In some embodiments of any ofthe above aspects, the method reduces adiposity of said subject. In someembodiments of any of the above aspects, the method reduces the weightsof epididymal and perirenal fat pads of said subject. In someembodiments of any of the above aspects, the method reduces the amountof subcutaneous and/or visceral fat of said subject. In some embodimentsof any of the above aspects, the method lowers the level of fastinginsulin of said subject. In some embodiments of any of the aboveaspects, the method lowers the level of blood glucose of said subject.In some embodiments of any of the above aspects, the method increasesinsulin sensitivity of said subject. In some embodiments of any of theabove aspects, the method increases the rate of glucose clearance ofsaid subject. In some embodiments of any of the above aspects, themethod improves the serum lipid profile of said subject. In someembodiments of any of the above aspects, the method does not reduce leanmass.

In some embodiments of any of the above aspects, the method increasesmuscle mass.

In some embodiments of any of the above aspects, the method reduces orinhibits the binding of activin and/or myostatin to their receptors.

In some embodiments of any of the above aspects, the polypeptide,nucleic acid, vector, or pharmaceutical composition is administered inan amount sufficient to increase muscle mass and/or strength, affectmyostatin, activin, and/or BMP9 signaling in the subject, or reduce orinhibit the binding of activin and/or myostatin to their receptors,

In some embodiments of any of the above aspects, the polypeptide,nucleic acid, vector, or pharmaceutical composition is administered inan amount sufficient to reduce body fat, reduce the amount ofsubcutaneous fat, reduce the amount of visceral fat, reduce adiposity,reduce the weights of epididymal and perirenal fat pads, reduce body fatpercentage, reduce body weight, reduce the percentage of body weightgain, reduce fasting insulin level, reduce blood glucose level, increaseinsulin sensitivity, affect myostatin, activin, and/or BMP9 signaling inthe subject, reduce the proliferation of adipose cells, reduce orinhibit the binding of activin and/or myostatin to their receptors,reduce LDL, reduce triglycerides, improve the serum lipid profile,regulate insulin biosynthesis and/or secretion from p-cells, delay,postpone, or reduce the need for insulin, or increase glucose clearance.

In some embodiments of any of the methods described herein, the methoddoes not cause a vascular complication (e.g., an increase vascularpermeability or leakage) in the subject. In some embodiments of any ofthe methods described herein, the method increases bone mineral densityin the subject.

In some embodiments of any of the above aspects, the variant has thesequence of SEQ ID NO: 69. In some embodiments, the variant having thesequence of SEQ ID NO: 69 has the amino acid K at position X₁₇, theamino acid sequence TEEN or TKEN at positions X₂₃, X₂₄, X₂₅, and X₂₆,and/or a C-terminal extension (e.g., 1, 2, 3, 4, 5, 6 or more additionalamino acids at the C-terminus, e.g., the amino acids NP or NPVTPK (SEQID NO: 155)). In some embodiments of any of the above aspects, themethod includes increasing muscle mass or treating a muscle disorder ina subject in need thereof (e.g., a subject having DMD, FSHD, IBM, ALS,sarcopenia, or cancer cachexia), affecting myostatin, activin, and/orBMP9 signaling in a subject (e.g., a subject having or at risk ofdeveloping DMD, FSHD, IBM, ALS, sarcopenia, cancer cachexia, obesity,Type-1 diabetes, or Type-2 diabetes), reducing body fat or body weightin a subject (e.g., a subject having obesity, Type-1 diabetes, or Type-2diabetes), or treating and/or preventing a metabolic disease in asubject (e.g., a subject having or at risk of developing obesity, Type-1diabetes, or Type-2 diabetes) by administering to the subject atherapeutically effective amount of a variant having the sequence of SEQID NO: 69, optionally having the amino acid K at position X₁₇, the aminoacid sequence TEEN or TKEN at positions X₂₃, X₂₄, X₂₅, and X₂₆, and/or aC-terminal extension, or a pharmaceutical composition containing saidvariant.

In some embodiments of any of the above aspects, the variant has thesequence of SEQ ID NO: 58. In some embodiments, the variant having thesequence of SEQ ID NO: 58 has the amino acid K at position X₁₇, theamino acid sequence TEEN or TKEN at positions X₂₃, X₂₄, X₂₅, and X₂₆,and/or a C-terminal extension (e.g., 1, 2, 3, 4, 5, 6 or more additionalamino acids at the C-terminus, e.g., the amino acids NP or NPVTPK (SEQID NO: 155)). In some embodiments of any of the above aspects, themethod includes increasing muscle mass or treating a muscle disorder ina subject in need thereof (e.g., a subject having DMD, FSHD, IBM, ALS,sarcopenia, or cancer cachexia), affecting myostatin, activin, and/orBMP9 signaling in a subject (e.g., a subject having or at risk ofdeveloping DMD, FSHD, IBM, ALS, sarcopenia, cancer cachexia, obesity,Type-1 diabetes, or Type-2 diabetes), reducing body fat or body weightin a subject (e.g., a subject having obesity, Type-1 diabetes, or Type-2diabetes), or treating and/or preventing a metabolic disease in asubject (e.g., a subject having or at risk of developing obesity, Type-1diabetes, or Type-2 diabetes) by administering to the subject atherapeutically effective amount of a variant having the sequence of SEQID NO: 58, optionally having the amino acid K at position X₁₇, the aminoacid sequence TEEN or TKEN at positions X₂₃, X₂₄, X₂₅, and X₂₆, and/or aC-terminal extension, or a pharmaceutical composition containing saidvariant.

In some embodiments of any of the above aspects, the variant has thesequence of SEQ ID NO: 6. In some embodiments, the variant having thesequence of SEQ ID NO: 6 has the amino acid K at position X₁₇, the aminoacid sequence TEEN or TKEN at positions X₂₃, X₂₄, X₂₅, and X₂₆, and/or aC-terminal extension (e.g., 1, 2, 3, 4, 5, 6, or more additional aminoacids at the C-terminus, e.g., the amino acids NP or NPVTPK (SEQ ID NO:155)). In some embodiments of any of the above aspects, the methodincludes increasing muscle mass or treating a muscle disorder in asubject in need thereof (e.g., a subject having DMD, FSHD, IBM, ALS,sarcopenia, or cancer cachexia), affecting myostatin, activin, and/orBMP9 signaling in a subject (e.g., a subject having or at risk ofdeveloping DMD, FSHD, IBM, ALS, sarcopenia, cancer cachexia, obesity,Type-1 diabetes, or Type-2 diabetes), reducing body fat or body weightin a subject (e.g., a subject having obesity, Type-1 diabetes, or Type-2diabetes), or treating and/or preventing a metabolic disease in asubject (e.g., a subject having or at risk of developing obesity, Type-1diabetes, or Type-2 diabetes) by administering to the subject atherapeutically effective amount of a variant having the sequence of SEQID NO: 6, optionally having the amino acid K at position X₁₇, the aminoacid sequence TEEN or TKEN at positions X₂₃, X₂₄, X₂₅, and X₂₆, and/or aC-terminal extension, or a pharmaceutical composition containing saidvariant.

In some embodiments of any of the above aspects, the variant has thesequence of SEQ ID NO: 38. In some embodiments, the variant having thesequence of SEQ ID NO: 38 has the amino acid K at position X₁₇, theamino acid sequence TEEN or TKEN at positions X₂₃, X₂₄, X₂₅, and X₂₆,and/or a C-terminal extension (e.g., 1, 2, 3, 4, 5, 6, or moreadditional amino acids at the C-terminus, e.g., the amino acids NP orNPVTPK (SEQ ID NO: 155)). In some embodiments of any of the aboveaspects, the method includes increasing muscle mass or treating a muscledisorder in a subject in need thereof (e.g., a subject having DMD, FSHD,IBM, ALS, sarcopenia, or cancer cachexia), affecting myostatin, activin,and/or BMP9 signaling in a subject (e.g., a subject having or at risk ofdeveloping DMD, FSHD, IBM, ALS, sarcopenia, cancer cachexia, obesity,Type-1 diabetes, or Type-2 diabetes), reducing body fat or body weightin a subject (e.g., a subject having obesity, Type-1 diabetes, or Type-2diabetes), or treating and/or preventing a metabolic disease in asubject (e.g., a subject having or at risk of developing obesity, Type-1diabetes, or Type-2 diabetes) by administering to the subject atherapeutically effective amount of a variant having the sequence of SEQID NO: 38, optionally having the amino acid K at position X₁₇, the aminoacid sequence TEEN or TKEN at positions X₂₃, X₂₄, X₂₅, and X₂₆, and/or aC-terminal extension, or a pharmaceutical composition containing saidvariant.

In some embodiments of any of the above aspects, the variant has thesequence of SEQ ID NO: 41. In some embodiments, the variant having thesequence of SEQ ID NO: 41 has the amino acid K at position X₁₇, theamino acid sequence TEEN or TKEN at positions X₂₃, X₂₄, X₂₅, and X₂₆,and/or a C-terminal extension (e.g., 1, 2, 3, 4, 5, 6, or moreadditional amino acids at the C-terminus, e.g., the amino acids NP orNPVTPK (SEQ ID NO: 155)). In some embodiments of any of the aboveaspects, the method includes increasing muscle mass or treating a muscledisorder in a subject in need thereof (e.g., a subject having DMD, FSHD,IBM, ALS, sarcopenia, or cancer cachexia), affecting myostatin, activin,and/or BMP9 signaling in a subject (e.g., a subject having or at risk ofdeveloping DMD, FSHD, IBM, ALS, sarcopenia, cancer cachexia, obesity,Type-1 diabetes, or Type-2 diabetes), reducing body fat or body weightin a subject (e.g., a subject having obesity, Type-1 diabetes, or Type-2diabetes), or treating and/or preventing a metabolic disease in asubject (e.g., a subject having or at risk of developing obesity, Type-1diabetes, or Type-2 diabetes) by administering to the subject atherapeutically effective amount of a variant having the sequence of SEQID NO: 41, optionally having the amino acid K at position X₁₇, the aminoacid sequence TEEN or TKEN at positions X₂₃, X₂₄, X₂₅, and X₂₆, and/or aC-terminal extension, or a pharmaceutical composition containing saidvariant.

In some embodiments of any of the above aspects, the variant has thesequence of SEQ ID NO: 44. In some embodiments, the variant having thesequence of SEQ ID NO: 44 has the amino acid K at position X₁₇, theamino acid sequence TEEN or TKEN at positions X₂₃, X₂₄, X₂₅, and X₂₆,and/or a C-terminal extension (e.g., 1, 2, 3, 4, 5, 6 or more additionalamino acids at the C-terminus, e.g., the amino acids NP or NPVTPK (SEQID NO: 155)). In some embodiments of any of the above aspects, themethod includes increasing muscle mass or treating a muscle disorder ina subject in need thereof (e.g., a subject having DMD, FSHD, IBM, ALS,sarcopenia, or cancer cachexia), affecting myostatin, activin, and/orBMP9 signaling in a subject (e.g., a subject having or at risk ofdeveloping DMD, FSHD, IBM, ALS, sarcopenia, cancer cachexia, obesity,Type-1 diabetes, or Type-2 diabetes), reducing body fat or body weightin a subject (e.g., a subject having obesity, Type-1 diabetes, or Type-2diabetes), or treating and/or preventing a metabolic disease in asubject (e.g., a subject having or at risk of developing obesity, Type-1diabetes, or Type-2 diabetes) by administering to the subject atherapeutically effective amount of a variant having the sequence of SEQID NO: 44, optionally having the amino acid K at position X₁₇, the aminoacid sequence TEEN or TKEN at positions X₂₃, X₂₄, X₂₅, and X₂₆, and/or aC-terminal extension, or a pharmaceutical composition containing saidvariant.

In some embodiments of any of the above aspects, the variant has thesequence of SEQ ID NO: 70. In some embodiments, the variant having thesequence of SEQ ID NO: 70 has the amino acid K at position X₁₇, theamino acid sequence TEEN or TKEN at positions X₂₃, X₂₄, X₂₅, and X₂₆,and/or a C-terminal extension (e.g., 1, 2, 3, 4, 5, 6 or more additionalamino acids at the C-terminus, e.g., the amino acids NP or NPVTPK (SEQID NO: 155)). In some embodiments of any of the above aspects, themethod includes increasing muscle mass or treating a muscle disorder ina subject in need thereof (e.g., a subject having DMD, FSHD, IBM, ALS,sarcopenia, or cancer cachexia), affecting myostatin, activin, and/orBMP9 signaling in a subject (e.g., a subject having or at risk ofdeveloping DMD, FSHD, IBM, ALS, sarcopenia, cancer cachexia, obesity,Type-1 diabetes, or Type-2 diabetes), reducing body fat or body weightin a subject (e.g., a subject having obesity, Type-1 diabetes, or Type-2diabetes), or treating and/or preventing a metabolic disease in asubject (e.g., a subject having or at risk of developing obesity, Type-1diabetes, or Type-2 diabetes) by administering to the subject atherapeutically effective amount of a variant having the sequence of SEQID NO: 70, optionally having the amino acid K at position X₁₇, the aminoacid sequence TEEN or TKEN at positions X₂₃, X₂₄, X₂₅, and X₂₆, and/or aC-terminal extension, or a pharmaceutical composition containing saidvariant.

In some embodiments of any of the above aspects, the variant has thesequence of SEQ ID NO: 71. In some embodiments, the variant having thesequence of SEQ ID NO: 71 has the amino acid K at position X₁₇, theamino acid sequence TEEN or TKEN at positions X₂₃, X₂₄, X₂₅, and X₂₆,and/or a C-terminal extension (e.g., 1, 2, 3, 4, 5, 6 or more additionalamino acids at the C-terminus, e.g., the amino acids VTPK). In someembodiments of any of the above aspects, the method includes increasingmuscle mass or treating a muscle disorder in a subject in need thereof(e.g., a subject having DMD, FSHD, IBM, ALS, sarcopenia, or cancercachexia), affecting myostatin, activin, and/or BMP9 signaling in asubject (e.g., a subject having or at risk of developing DMD, FSHD, IBM,ALS, sarcopenia, cancer cachexia, obesity, Type-1 diabetes, or Type-2diabetes), reducing body fat or body weight in a subject (e.g., asubject having obesity, Type-1 diabetes, or Type-2 diabetes), ortreating and/or preventing a metabolic disease in a subject (e.g., asubject having or at risk of developing obesity, Type-1 diabetes, orType-2 diabetes) by administering to the subject a therapeuticallyeffective amount of a variant having the sequence of SEQ ID NO: 71,optionally having the amino acid K at position X₁₇, the amino acidsequence TEEN or TKEN at positions X₂₃, X₂₄, X₂₅, and X₂₆, and/or aC-terminal extension, or a pharmaceutical composition containing saidvariant.

In some embodiments of any of the above aspects, the variant has thesequence of SEQ ID NO: 72. In some embodiments, the variant having thesequence of SEQ ID NO: 72 has the amino acid K at position X₁₇ and/orthe amino acid sequence TEEN or TKEN at positions X₂₃, X₂₄, X₂₅, andX₂₆. In some embodiments of any of the above aspects, the methodincludes increasing muscle mass or treating a muscle disorder in asubject in need thereof (e.g., a subject having DMD, FSHD, IBM, ALS,sarcopenia, or cancer cachexia), affecting myostatin, activin, and/orBMP9 signaling in a subject (e.g., a subject having or at risk ofdeveloping DMD, FSHD, IBM, ALS, sarcopenia, cancer cachexia, obesity,Type-1 diabetes, or Type-2 diabetes), reducing body fat or body weightin a subject (e.g., a subject having obesity, Type-1 diabetes, or Type-2diabetes), or treating and/or preventing a metabolic disease in asubject (e.g., a subject having or at risk of developing obesity, Type-1diabetes, or Type-2 diabetes) by administering to the subject atherapeutically effective amount of a variant having the sequence of SEQID NO: 72, optionally having the amino acid K at position X₁₇ and/or theamino acid sequence TEEN or TKEN at positions X₂₃, X₂₄, X₂₅, and X₂₆.

Definitions

As used herein, the term “extracellular activin receptor type IIa(ActRIIa) variant” refers to a peptide including a soluble,extracellular portion of the single transmembrane receptor, ActRIIa,that has at least one amino acid substitution relative to a wild-typeextracellular ActRIIa (e.g., bold portion of the sequence of SEQ ID NO:75 shown below) or an extracellular ActRIIa having any one of thesequences of SEQ ID NOs: 76-96. The sequence of the wild-type, humanActRIIa precursor protein is shown below (SEQ ID NO: 75), in which thesignal peptide is italicized and the extracellular portion is bold.

Wild-type, human ActRIIa precursor protein (SEQ ID NO: 75):MGAAAKLAFAVFLISCSS GAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPPYYNILLYSLVPLMLIAGIVICAFWVYRHHKMAYPPVLVPTQDPGPPPPSPLLGLKPLQLLEVKARGRFGCVWKAQLLNEYVAVKIFPIQDKQSWQNEYEVYSLPGMKHENILQFIGAEKRGTSVDVDLWLITAFHEKGSLSDFLKANVVSWNELCHIAETMARGLAYLHEDIPGLKDGHKPAISHRDIKSKNVLLKNNLTACIADFGLALKFEAGKSAGDTHGQVGTRRYMAPEVLEGAINFQRDAFLRIDMYAMGLVLWELASRCTAADGPVDEYMLPFEEEIGQHPSLEDMQEVVVHKKKRPVLRDYWQKHAGMAMLCETIEECWDHDAEARLSAGCVGERITQMQRLTNIITTEDIVTVVTMVTNVDFPPKESSL

An extracellular ActRIIa variant may have a sequence of any one of SEQID NOs: 1-72. In particular embodiments, an extracellular ActRIIavariant has a sequence of any one of SEQ ID NOs: 6-72 (Table 2). In someembodiments, an extracellular ActRIIa variant may have at least 85%(e.g., at least 85%, 87%, 90%, 92%, 95%, 97%, or greater) amino acidsequence identity to the sequence of a wild-type extracellular ActRIIa(SEQ ID NO: 73).

As used herein, the term “extracellular ActRIIb variant” refers to apeptide including a soluble, extracellular portion of the singletransmembrane receptor, ActRIIb, that has at least one amino acidsubstitution relative to a wild-type extracellular ActRIIb (e.g., thesequence of SEQ ID NO: 74). An extracellular ActRIIb variant may havethe sequence of SEQ ID NO: 149 shown below:

extracellular ActRIIb variant (SEQ ID NO: 149):GRGEAETRECIFYNANWEKDRTNQSGLEPCYGDQDKRRHCFASWKNSSGTIELVKQGCWLDDINCYDRQECVAKKDSPEVYFCCCEGNFCNERFTHLP EAGGPEVTYEPPPTAPT

As used herein, the term “linker” refers to a linkage between twoelements, e.g., peptides or protein domains. A polypeptide describedherein may include an extracellular ActRIIa variant (e.g., anextracellular ActRIIa variant having a sequence of any one of SEQ IDNOs: 1-72 (e.g., SEQ ID NOs: 6-72)) fused to a moiety. The moiety mayincrease stability or improve pharmacokinetic properties of thepolypeptide. The moiety (e.g., Fc domain monomer, a wild-type Fc domain,an Fc domain with amino acid substitutions (e.g., one or moresubstitutions that reduce dimerization), an albumin-binding peptide, afibronectin domain, or a human serum albumin) may be fused to thepolypeptide by way of a linker. A linker can be a covalent bond or aspacer. The term “bond” refers to a chemical bond, e.g., an amide bondor a disulfide bond, or any kind of bond created from a chemicalreaction, e.g., chemical conjugation. The term “spacer” refers to amoiety (e.g., a polyethylene glycol (PEG) polymer) or an amino acidsequence (e.g., a 1-200 amino acid sequence) occurring between twoelements, e.g., peptides or protein domains, to provide space and/orflexibility between the two elements. An amino acid spacer is part ofthe primary sequence of a polypeptide (e.g., fused to the spacedpeptides via the polypeptide backbone). The formation of disulfidebonds, e.g., between two hinge regions that form an Fc domain, is notconsidered a linker.

As used herein, the term “Fc domain” refers to a dimer of two Fc domainmonomers. An Fc domain has at least 80% sequence identity (e.g., atleast 85%, 90%, 95%, 97%, or 100% sequence identity) to a human Fcdomain that includes at least a C_(H) ² domain and a C_(H) ³ domain. AnFc domain monomer includes second and third antibody constant domains(C_(H) ² and C_(H) ³). In some embodiments, the Fc domain monomer alsoincludes a hinge domain. An Fc domain does not include any portion of animmunoglobulin that is capable of acting as an antigen-recognitionregion, e.g., a variable domain or a complementarity determining region(CDR). In the wild-type Fc domain, the two Fc domain monomers dimerizeby the interaction between the two C_(H) ³ antibody constant domains, aswell as one or more disulfide bonds that form between the hinge domainsof the two dimerizing Fc domain monomers. In some embodiments, an Fcdomain may be mutated to lack effector functions, typical of a “dead Fcdomain.” In certain embodiments, each of the Fc domain monomers in an Fcdomain includes amino acid substitutions in the C_(H) ² antibodyconstant domain to reduce the interaction or binding between the Fcdomain and an Fcγ receptor. In some embodiments, the Fc domain containsone or more amino acid substitutions that reduce or inhibit Fc domaindimerization. An Fc domain can be any immunoglobulin antibody isotype,including IgG, IgE, IgM, IgA, or IgD. Additionally, an Fc domain can bean IgG subtype (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4). The Fc domaincan also be a non-naturally occurring Fc domain, e.g., a recombinant Fcdomain.

As used herein, the term “albumin-binding peptide” refers to an aminoacid sequence of 12 to 16 amino acids that has affinity for andfunctions to bind serum albumin. An albumin-binding peptide can be ofdifferent origins, e.g., human, mouse, or rat. In some embodiments, analbumin-binding peptide has the sequence DICLPRWGCLW (SEQ ID NO: 152).

As used herein, the term “fibronectin domain” refers to a high molecularweight glycoprotein of the extracellular matrix, or a fragment thereof,that binds to, e.g., membrane-spanning receptor proteins such asintegrins and extracellular matrix components such as collagens andfibrins. In some embodiments, a fibronectin domain is a fibronectin typeIII domain (SEQ ID NO: 153) having amino acids 610-702 of the sequenceof UniProt ID NO: P02751. In other embodiments, a fibronectin domain isan adnectin protein.

As used herein, the term “human serum albumin” refers to the albuminprotein present in human blood plasma. Human serum albumin is the mostabundant protein in the blood. It constitutes about half of the bloodserum protein. In some embodiments, a human serum albumin has thesequence of UniProt ID NO: P02768 (SEQ ID NO: 154).

As used herein, the term “fused” is used to describe the combination orattachment of two or more elements, components, or protein domains,e.g., peptides or polypeptides, by means including chemical conjugation,recombinant means, and chemical bonds, e.g., amide bonds. For example,two single peptides in tandem series can be fused to form one contiguousprotein structure, e.g., a polypeptide, through chemical conjugation, achemical bond, a peptide linker, or any other means of covalent linkage.In some embodiments of a polypeptide described herein, an extracellularActRIIa variant (e.g., an extracellular ActRIIa variant having thesequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) may befused in tandem series to the N- or C-terminus of a moiety (e.g., Fcdomain monomer (e.g., the sequence of SEQ ID NO: 97) a wild-type Fcdomain (e.g., the sequence of SEQ ID NO: 151), an Fc domain with aminoacid substitutions (e.g., one or more substitutions that reducedimerization), an albumin-binding peptide (e.g., the sequence of SEQ IDNO: 152), a fibronectin domain (e.g., the sequence of SEQ ID NO: 153),or a human serum albumin (e.g., the sequence of SEQ ID NO: 154)) by wayof a linker. For example, an extracellular ActRIIa variant is fused to amoiety (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domainwith amino acid substitutions (e.g., one or more substitutions thatreduce dimerization), an albumin-binding peptide, a fibronectin domain,or a human serum albumin) by way of a peptide linker, in which theN-terminus of the peptide linker is fused to the C-terminus of theextracellular ActRIIa variant through a chemical bond, e.g., a peptidebond, and the C-terminus of the peptide linker is fused to theN-terminus of the moiety (e.g., Fc domain monomer, wild-type Fc domain,Fc domain with amino acid substitutions (e.g., one or more substitutionsthat reduce dimerization), albumin-binding peptide, fibronectin domain,or human serum albumin) through a chemical bond, e.g., a peptide bond.

As used herein, the term “C-terminal extension” refers to the additionof one or more amino acids to the C-terminus of a polypeptide includingan extracellular ActRIIa variant (e.g., an extracellular ActRIIa varianthaving the sequence of any one of SEQ ID NOs: 1-70 (e.g., SEQ ID NOs:6-70)). The C-terminal extension can be 1-6 amino acids (e.g., 1, 2, 3,4, 5, 6 or more amino acids). Exemplary C-terminal extensions are theamino acid sequence NP (a two amino acid C-terminal extension) and theamino acid sequence NPVTPK (SEQ ID NO: 155) (a six amino acid C-terminalextension). Any amino acid sequence that does not disrupt the activityof the polypeptide can be used. SEQ ID NO: 71, which is the sequence ofSEQ ID NO: 69 with a C-terminal extension of NP, and SEQ ID NO: 72,which is the sequence of SEQ ID NO: 69 with a C-terminal extension ofNPVTPK (SEQ ID NO: 155), represent two of the possible ways that apolypeptide of the invention can be modified to include a C-terminalextension.

As used herein, the term “percent (%) identity” refers to the percentageof amino acid (or nucleic acid) residues of a candidate sequence, e.g.,an extracellular ActRIIa variant, that are identical to the amino acid(or nucleic acid) residues of a reference sequence, e.g., a wild-typeextracellular ActRIIa (e.g., SEQ ID NO: 73), after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent identity (i.e., gaps can be introduced in one or both of thecandidate and reference sequences for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes).Alignment for purposes of determining percent identity can be achievedin various ways that are within the skill in the art, for instance,using publicly available computer software such as BLAST, ALIGN, orMegalign (DNASTAR) software. Those skilled in the art can determineappropriate parameters for measuring alignment, including any algorithmsneeded to achieve maximal alignment over the full length of thesequences being compared. In some embodiments, the percent amino acid(or nucleic acid) sequence identity of a given candidate sequence to,with, or against a given reference sequence (which can alternatively bephrased as a given candidate sequence that has or includes a certainpercent amino acid (or nucleic acid) sequence identity to, with, oragainst a given reference sequence) is calculated as follows:100×(fraction of A/B)where A is the number of amino acid (or nucleic acid) residues scored asidentical in the alignment of the candidate sequence and the referencesequence, and where B is the total number of amino acid (or nucleicacid) residues in the reference sequence. In some embodiments where thelength of the candidate sequence does not equal to the length of thereference sequence, the percent amino acid (or nucleic acid) sequenceidentity of the candidate sequence to the reference sequence would notequal to the percent amino acid (or nucleic acid) sequence identity ofthe reference sequence to the candidate sequence.

In particular embodiments, a reference sequence aligned for comparisonwith a candidate sequence may show that the candidate sequence exhibitsfrom 50% to 100% identity across the full length of the candidatesequence or a selected portion of contiguous amino acid (or nucleicacid) residues of the candidate sequence. The length of the candidatesequence aligned for comparison purpose is at least 30%, e.g., at least40%, e.g., at least 50%, 60%, 70%, 80%, 90%, or 100% of the length ofthe reference sequence. When a position in the candidate sequence isoccupied by the same amino acid (or nucleic acid) residue as thecorresponding position in the reference sequence, then the molecules areidentical at that position.

As used herein, the term “serum half-life” refers to, in the context ofadministering a therapeutic protein to a subject, the time required forplasma concentration of the protein in the subject to be reduced byhalf. The protein can be redistributed or cleared from the bloodstream,or degraded, e.g., by proteolysis. As described herein, a polypeptideincluding an extracellular ActRIIa variant (e.g., an extracellularActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g.,SEQ ID NOs: 6-72)) displays a serum half-life of 7 days in humans.

As used herein, the term “metabolic disease” refers to a disease,disorder, or syndrome that is related to a subject's metabolism, such asbreaking down carbohydrates, proteins, and fats in food to releaseenergy, and converting chemicals into other substances and transportingthem inside cells for energy utilization and/or storage. Some symptomsof a metabolic disease include high serum triglycerides, highlow-density cholesterol (LDL), low high-density cholesterol (HDL),and/or high fasting insulin levels, elevated fasting plasma glucose,abdominal (central) obesity, and elevated blood pressure. Metabolicdiseases increase the risk of developing other diseases, such ascardiovascular disease. In the present invention, metabolic diseasesinclude, but are not limited to, obesity, Type-1 diabetes, and Type-2diabetes.

As used herein, the term “percentage of body weight gain” refers to thepercentage of gained body weight compared to a prior body weight of asubject at a prior time. The percentage of body weight gain can becalculated as follows:100×[(body weight at a later time−body weight at a prior time)/(bodyweight at a prior time)]In the present invention, administration of a polypeptide including anextracellular ActRIIa variant (e.g., an extracellular ActRIIa varianthaving the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs:6-72)), a nucleic acid molecule encoding a polypeptide including anextracellular ActRIIa variant (e.g., an extracellular ActRIIa varianthaving the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs:6-72)), or vector containing such a nucleic acid molecule to a subjectreduces the percentage of body weight gain of the subject.

As used herein, the term “appetite for food intake” refers to asubject's natural desire or need for food. The appetite for food intakeof a subject can be monitored by measuring the amount of food consumedafter the polypeptide including an extracellular ActRIIa variant (e.g.,an extracellular ActRIIa variant having the sequence of any one of SEQID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) is administered. In the presentinvention, administration of a polypeptide including an extracellularActRIIa variant (e.g., an extracellular ActRIIa variant having thesequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)), anucleic acid molecule encoding a polypeptide including an extracellularActRIIa variant (e.g., an extracellular ActRIIa variant having thesequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)), orvector containing such a nucleic acid molecule to a subject does notaffect the subject's appetite for food intake.

As used herein, the term “adiposity” refers to the fat stored in theadipose tissue of a subject. In the present invention, administration ofa polypeptide including an extracellular ActRIIa variant (e.g., anextracellular ActRIIa variant having the sequence of any one of SEQ IDNOs: 1-72 (e.g., SEQ ID NOs: 6-72)), a nucleic acid molecule encoding apolypeptide including an extracellular ActRIIa variant (e.g., anextracellular ActRIIa variant having the sequence of any one of SEQ IDNOs: 1-72 (e.g., SEQ ID NOs: 6-72)), or vector containing such a nucleicacid molecule to a subject reduces the subject's adiposity withoutaffecting lean mass.

As used herein, the term “lean mass” refers to a component of bodycomposition which includes, e.g., lean mass, body fat, and body fluid.Normally lean mass is calculated by subtracting the weights of body fatand body fluid from total body weight. Typically, a subject's lean massis between 60% and 90% of totally body weight. In the present invention,administration of a polypeptide including an extracellular ActRIIavariant (e.g., an extracellular ActRIIa variant having the sequence ofany one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)), a nucleic acidmolecule encoding a polypeptide including an extracellular ActRIIavariant (e.g., an extracellular ActRIIa variant having the sequence ofany one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)), or vectorcontaining such a nucleic acid molecule to a subject reduces thesubject's adiposity (i.e., fat) without affecting lean mass.

As used herein, the term “epididymal and perirenal fat pads” refers tothe tightly packed fat cells in the epididymis and around the kidney. Inthe present invention, administration of a polypeptide including anextracellular ActRIIa variant (e.g., an extracellular ActRIIa varianthaving the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs:6-72)), a nucleic acid molecule encoding a polypeptide including anextracellular ActRIIa variant (e.g., an extracellular ActRIIa varianthaving the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs:6-72)), or vector containing such a nucleic acid molecule to a subjectreduces the weights of epididymal and perirenal fat pads of the subject.

As used herein, the term “fasting insulin” refers to a subject's levelof insulin while the subject has not had any food intake for a length oftime (i.e., 12-24 hours). Fasting insulin level is used in diagnosingmetabolic diseases. Fasting insulin level is also used as an indicationof whether a subject is at the risk of developing a metabolic disease.Normally, in a subject suffering from Type-1 diabetes, the subject'sfasting insulin level is low compared to that of a healthy subject. In asubject suffering from insulin resistance (i.e., Type-2 diabetes), thesubject's fasting insulin level is high compared to that of a healthysubject. In the present invention, administration of a polypeptideincluding an extracellular ActRIIa variant (e.g., an extracellularActRIIa variant having the sequence of any one of SEQ ID NOs: 1-72(e.g., SEQ ID NOs: 6-72)), a nucleic acid molecule encoding apolypeptide including an extracellular ActRIIa variant (e.g., anextracellular ActRIIa variant having the sequence of any one of SEQ IDNOs: 1-72 (e.g., SEQ ID NOs: 6-72)), or vector containing such a nucleicacid molecule to a subject lowers the subject's fasting insulin level.

As used herein, the term “rate of glucose clearance” refers to the rateat which glucose is being cleared from the blood. The rate of glucoseclearance can be measured in a glucose tolerance test (GTT). In a GTT, asubject is given a certain amount of glucose and blood samples are takenafterward to determine how quickly it is cleared from the blood. Therate of glucose clearance can be used as a parameter in diagnosingand/or determining the risk of developing metabolic diseases such asobesity, diabetes, and insulin resistance.

As used herein, the term “serum lipid profile” refers to the measurementof the distribution of different types of lipids and lipoproteins in asubject's serum. Such measurement can be accomplished by a panel ofblood tests. The types of lipids and lipoproteins in a subject's seruminclude, but are not limited to, cholesterol (e.g., high-densitylipoprotein (HDL) and low-density lipoprotein (LDL)), triglyceride, andfree fatty acid (FFA). The distribution of the different types of lipidsand lipoproteins can be used as a parameter in diagnosing and/ordetermining the risk of developing metabolic diseases such as obesity,diabetes, and insulin resistance. High levels of cholesterol, especiallylow-density lipoprotein, is generally regarded as an indication or riskfactor for developing certain metabolic diseases, or in some severemedical cases, cardiovascular diseases. In the present invention,administration of a polypeptide including an extracellular ActRIIavariant (e.g., an extracellular ActRIIa variant having the sequence ofany one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)), a nucleic acidmolecule encoding a polypeptide including an extracellular ActRIIavariant (e.g., an extracellular ActRIIa variant having the sequence ofany one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)), or vectorcontaining such a nucleic acid molecule to a subject improves thesubject's serum lipid profile such that the levels of cholesterol(especially low-density lipoprotein) and triglyceride are lowered.

As used herein, the term “affinity” or “binding affinity” refers to thestrength of the binding interaction between two molecules. Generally,binding affinity refers to the strength of the sum total of non-covalentinteractions between a molecule and its binding partner, such as anextracellular ActRIIa variant and BMP9 or activin A. Unless indicatedotherwise, binding affinity refers to intrinsic binding affinity, whichreflects a 1:1 interaction between members of a binding pair. Thebinding affinity between two molecules is commonly described by thedissociation constant (K_(D)) or the affinity constant (K_(A)). Twomolecules that have low binding affinity for each other generally bindslowly, tend to dissociate easily, and exhibit a large K_(D). Twomolecules that have high affinity for each other generally bind readily,tend to remain bound longer, and exhibit a small K_(D). The K_(D) of twointeracting molecules may be determined using methods and techniqueswell known in the art, e.g., surface plasmon resonance. K_(D) iscalculated as the ratio of k_(on)/k_(on).

As used herein, the term “muscle mass” refers to a component of bodycomposition. Normally muscle mass is calculated by subtracting theweights of body fat and body fluid from total body weight.

The percentage of muscle mass may vary greatly among individualsdepending on a subject's genetic makeup, age, race, and health status,etc. Typically, a subject's muscle mass may be between 20% and 50% oftotally body weight.

As used herein, the phrase “affecting myostatin, activin, and/or BMP9signaling” means changing the binding of myostatin, activin, and/or BMP9to their receptors, e.g., ActRIIa, ActRIIb, and BMPRII (e.g., ActRIIa).In some embodiments, a polypeptide including an extracellular ActRIIavariant described herein reduces or inhibits the binding of myostatin,activin, and/or BMP9 to their receptors, e.g., ActRIIa, ActRIIb, andBMPRII (e.g., ActRIIa). As described herein, a polypeptide of theinvention including an extracellular ActRIIa variant (e.g., anextracellular ActRIIa variant having the sequence of any one of SEQ IDNOs: 1-72 (e.g., SEQ ID NOs: 6-72)) may have weak binding affinity toBMP9 (e.g., K_(D) of 200 pM or higher).

As used herein, the term “vascular complication” refers to a vasculardisorder or any damage to the blood vessels, such as damage to the bloodvessel walls. Damage to the blood vessel walls may cause an increase invascular permeability or leakage. The term “vascular permeability orleakage” refers to the capacity of the blood vessel walls to allow theflow of small molecules, proteins, and cells in and out of bloodvessels. An increase in vascular permeability or leakage may be causedby an increase in the gaps (e.g., an increase in the size and/or numberof the gaps) between endothelial cells that line the blood vessel wallsand/or thinning of the blood vessel walls.

As used herein, the term “polypeptide” describes a single polymer inwhich the monomers are amino acid residues which are covalentlyconjugated together through amide bonds. A polypeptide is intended toencompass any amino acid sequence, either naturally occurring,recombinant, or synthetically produced.

As used herein, the term “homodimer” refers to a molecular constructformed by two identical macromolecules, such as proteins or nucleicacids. The two identical monomers may form a homodimer by covalent bondsor non-covalent bonds. For example, an Fc domain may be a homodimer oftwo Fc domain monomers if the two Fc domain monomers contain the samesequence. In another example, a polypeptide described herein includingan extracellular ActRIIa variant fused to an Fc domain monomer may forma homodimer through the interaction of two Fc domain monomers, whichform an Fc domain in the homodimer.

As used herein, the term “heterodimer” refers to a molecular constructformed by two different macromolecules, such as proteins or nucleicacids. The two monomers may form a heterodimer by covalent bonds ornon-covalent bonds. For example, a polypeptide described hereinincluding an extracellular ActRIIa variant fused to an Fc domain monomermay form a heterodimer through the interaction of two Fc domainmonomers, each fused to a different ActRIIa variant, which form an Fcdomain in the heterodimer.

As used herein, the term “host cell” refers to a vehicle that includesthe necessary cellular components, e.g., organelles, needed to expressproteins from their corresponding nucleic acids. The nucleic acids aretypically included in nucleic acid vectors that can be introduced intothe host cell by conventional techniques known in the art(transformation, transfection, electroporation, calcium phosphateprecipitation, direct microinjection, etc.). A host cell may be aprokaryotic cell, e.g., a bacterial cell, or a eukaryotic cell, e.g., amammalian cell (e.g., a CHO cell or a HEK293 cell).

As used herein, the term “therapeutically effective amount” refers anamount of a polypeptide, nucleic acid, or vector of the invention or apharmaceutical composition containing a polypeptide, nucleic acid, orvector of the invention effective in achieving the desired therapeuticeffect in treating a patient having a disease, such as a muscle disease,or a condition involving weakness and atrophy of muscles, e.g., Duchennemuscular dystrophy (DMD), facioscapulohumeral muscular dystrophy (FSHD),inclusion body myositis (IBM), amyotrophic lateral sclerosis (ALS),sarcopenia, or cancer cachexia. The term “therapeutically effectiveamount” also refers an amount of a polypeptide, nucleic acid, or vectorof the invention or a pharmaceutical composition containing apolypeptide, nucleic acid, or vector of the invention effective inachieving the desired therapeutic effect in treating a patient having adisease, such as a metabolic disease, or a condition involving excessbody weight, excess body fat, high blood glucose, high fasting insulinlevels, or insulin resistance, e.g., obesity, Type-1 diabetes, or Type-2diabetes. In particular, the therapeutically effective amount of thepolypeptide, nucleic acid, or vector avoids adverse side effects.

As used herein, the term “pharmaceutical composition” refers to amedicinal or pharmaceutical formulation that includes an activeingredient as well as excipients and diluents to enable the activeingredient suitable for the method of administration. The pharmaceuticalcomposition of the present invention includes pharmaceuticallyacceptable components that are compatible with the polypeptide, nucleicacid, or vector. The pharmaceutical composition may be in tablet orcapsule form for oral administration or in aqueous form for intravenousor subcutaneous administration.

As used herein, the term “pharmaceutically acceptable carrier orexcipient” refers to an excipient or diluent in a pharmaceuticalcomposition. The pharmaceutically acceptable carrier must be compatiblewith the other ingredients of the formulation and not deleterious to therecipient. In the present invention, the pharmaceutically acceptablecarrier or excipient must provide adequate pharmaceutical stability tothe polypeptide including an extracellular ActRIIa variant, the nucleicacid molecule(s) encoding the polypeptide, or a vector containing suchnucleic acid molecule(s). The nature of the carrier or excipient differswith the mode of administration. For example, for intravenousadministration, an aqueous solution carrier is generally used; for oraladministration, a solid carrier is preferred.

As used herein, the term “treating and/or preventing” refers to thetreatment and/or prevention of a disease, e.g., a metabolic disease(e.g., obesity, Type1 and Type-2 diabetes) or a muscle disease (e.g.,DMD, FSHD, IBM, and ALS), using methods and compositions of theinvention. Generally, treating a metabolic or muscle disease occursafter a subject has developed the metabolic or muscle disease and/or isalready diagnosed with the metabolic or muscle disease. Preventing ametabolic or muscle disease refers to steps or procedures taken when asubject is at risk of developing the metabolic or muscle disease. Thesubject may show signs or mild symptoms that are judged by a physicianto be indications or risk factors for developing the metabolic or muscledisease or have a family history or genetic predisposition of developingthe metabolic or muscle disease, but has not yet developed the disease.

As used herein, the term “subject” refers to a mammal, e.g., preferablya human. Mammals include, but are not limited to, humans and domesticand farm animals, such as monkeys (e.g., a cynomolgus monkey), mice,dogs, cats, horses, and cows, etc.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a sequence alignment showing the wild-type sequences ofextracellular ActRIIa and ActRIIb and the amino acid substitutions inActRIIa variants.

FIGS. 2A and 2B are scatter plots showing the effects of extracellularActRIIa variants on body weight. Mice received a single hydrodynamicinjection of a plasmid construct encoding the indicated ActRIIa variantor a control plasmid.

FIGS. 3A and 3B are bar graphs showing the effects of extracellularActRIIa variants on muscle mass.

FIG. 4A is a scatter plot showing the effects of extracellular ActRIIavariants on body weight. Mice received an intraperitoneal injection ofthe indicated purified recombinant ActRIIa variant or a vehicle controltwice weekly for four weeks.

FIG. 4B is a bar graph showing the effects of extracellular ActRIIavariants on individual muscle weights by tissue analysis.

FIG. 5A is a scatter plot showing the effects of extracellular ActRIIavariants on body weight during the course of the study. Mice received asingle hydrodynamic injection of a plasmid construct encoding theindicated ActRIIa variant or a control plasmid.

FIG. 5B is a bar graph showing the effects of extracellular ActRIIavariants on body weight at the end of 28 days.

FIGS. 6A and 6B are bar graphs showing the effects of extracellularActRIIa variants on body weight by tissue analysis.

FIGS. 7A and 7B are scatter plots showing the effects of different dosesof extracellular ActRIIa variants on body weight. Mice received anintraperitoneal injection of the indicated purified recombinant ActRIIavariant or a vehicle control twice weekly for four weeks.

FIGS. 8A and 8B are bar graphs showing the effects of different doses ofextracellular ActRIIa variants on muscle mass (FIG. 8A) and fat mass(FIG. 8B).

FIGS. 9A and 9B are bar graphs showing the effects of different doses ofextracellular ActRIIa variants on muscle weights by tissue analysis.

DETAILED DESCRIPTION OF THE INVENTION

The invention features polypeptides that include an extracellularactivin receptor type IIa (ActRIIa) variant. In some embodiments, apolypeptide of the invention includes an extracellular ActRIIa variantfused to a moiety (e.g., Fc domain monomer, a wild-type Fc domain, an Fcdomain with amino acid substitutions (e.g., one or more substitutionsthat reduce dimerization), an albumin-binding peptide, a fibronectindomain, or a human serum albumin). A polypeptide including anextracellular ActRIIa variant fused to an Fc domain monomer may alsoform a dimer (e.g., homodimer or heterodimer) through the interactionbetween two Fc domain monomers. The ActRIIa variants described hereinhave weak binding affinity or no binding affinity to bone morphogeneticprotein 9 (BMP9) compared to activins and myostatin. The invention alsoincludes methods of treating diseases and conditions involving weaknessand atrophy of muscles by increasing muscle mass and strength, methodsof treating or preventing metabolic diseases, or methods of affectingmyostatin, activin, and/or BMP9 signaling in a subject by administeringto the subject a polypeptide including an extracellular ActRIIa variantdescribed herein. 1. Extracellular activin receptor type IIa (ActRIIa)variants Activin type II receptors are single transmembrane domainreceptors that modulate signals for ligands in the transforming growthfactor β (TGF-β) superfamily. Ligands in the TGF-β superfamily areinvolved in a host of physiological processes, such as muscle growth,vascular growth, cell differentiation, homeostasis, and osteogenesis.Examples of ligands in the TGF-β superfamily include, e.g., activin,inhibin, growth differentiation factors (GDFs) (e.g., GDF8, also knownas myostatin), and bone morphogenetic proteins (BMPs) (e.g., BMP9).Myostatin and activins are known to play a role in the regulation ofskeletal muscle growth. For example, mice without myostatin show a largeincrease in skeletal muscle mass.

Activins are also highly expressed in adipose tissue, and increasedmyostatin levels and activin receptor levels have been observed insubcutaneous and visceral fat of obese mice. Additionally, myostatin hasbeen shown to be elevated in skeletal muscle and plasma of obese andinsulin resistant women, and both type I and type II activin receptorshave been linked to pancreatic function and diabetes. These data suggestthat increased signaling through activin receptors, either due toincreased expression of activin ligands (e.g., activin, myostatin) orincreased expression of activin receptors themselves, could lead toobesity and metabolic disorders, such as Type-1 and Type-2 diabetes.Methods that reduce or inhibit this signaling could, therefore, be usedin the treatment of obesity and metabolic disorders.

There exist two types of activin type II receptors: ActRIIa and ActRIIb.Studies have shown that BMP9 binds ActRIIb with about 300-fold higherbinding affinity than ActRIIa (see, e.g., Townson et al., J. Biol. Chem.287:27313, 2012). ActRIIa is known to have a longer half-life comparedto ActRIIb. The present invention describes extracellular ActRIIavariants that are constructed by introducing amino acid residues ofActRIIb to ActRIIa, with the goal of imparting physiological propertiesconferred by ActRIIb, while also maintaining beneficial physiologicaland pharmacokinetic properties of ActRIIa. The optimum peptides confersignificant increases in muscle mass, while retaining longer serumhalf-life and low binding-affinity to BMP9, for example. The preferredActRIIa variants also exhibit improved binding to activins and/ormyostatin compared to wild-type ActRIIa, which allows them to competewith endogenous activin receptors for ligand binding and reduce orinhibit endogenous activin receptor signaling. These variants can beused to treat disorders in which activin receptor signaling is elevated,such as metabolic disorders, leading to a reduction in body fat, bodyweight, or insulin resistance (e.g., an increase in insulinsensitivity). In some embodiments, amino acid substitutions may beintroduced to an extracellular ActRIIa variant to reduce or remove thebinding affinity of the variant to BMP9. The wild-type amino acidsequences of the extracellular portions of human ActRIIa and ActRIIb areshown below.

Human ActRIIa, extracellular portion (SEQ ID NO: 73):GAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSY FPEMEVTQPTSHuman ActRIIb, extracellular portion (SEQ ID NO: 74):GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLP EAGGPEVTYEPPPTAPT

Polypeptides described herein include an extracellular ActRIIa varianthaving at least one amino acid substitution relative to the wild-typeextracellular ActRIIa having the sequence of SEQ ID NO: 73 or theextracellular ActRIIa having any one of the sequences of SEQ ID NOs:76-96. Possible amino acid substitutions at 27 different positions maybe introduced to an extracellular ActRIIa variant (Table 1). In someembodiments, an extracellular ActRIIa variant may have at least 85%(e.g., at least 85%, 87%, 90%, 92%, 95%, 97%, or greater) amino acidsequence identity to the sequence of a wild-type extracellular ActRIIa(SEQ ID NO: 73). An extracellular ActRIIa variant may have one or more(e.g., 1-27, 1-25, 1-23, 1-21, 1-19, 1-17, 1-15, 1-13, 1-11, 1-9, 1-7,1-5, 1-3, or 1-2; e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) amino acidsubstitutions relative the sequence of a wild-type extracellular ActRIIa(SEQ ID NO: 73). In some embodiments, an extracellular ActRIIa variant(e.g., an extracellular ActRIIa variant having a sequence of SEQ IDNO: 1) may include amino acid substitutions at all of the 27 positionsas listed in Table 1. In some embodiments, an extracellular ActRIIavariant may include amino acid substitutions at a number of positions,e.g., at 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26 out of the 27positions, as listed in Table 1.

Amino acid substitutions can worsen or improve the activity and/orbinding affinity of the ActRIIa variants of the invention. To maintainpolypeptide function, it is important that the lysine (K) at positionX₁₇ in the sequences shown in Tables 1 and 2 (SEQ ID NOs: 1-72 (e.g.,SEQ ID NOs: 6-72)) be retained. Substitutions at that position can leadto a loss of activity. For example, an ActRIIa variant having thesequenceGAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCYATWRNISGSIEIVAKGCWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 150) has reducedactivity in vivo, indicating that the substitution of alanine (A) forlysine (K) at X₁₇ is not tolerated. ActRIIa variants of the invention,including variants in Tables 1 and 2 (e.g., SEQ ID NOs: 1-72 (e.g., SEQID NOs: 6-72), therefore, retain amino acid K at position X₁₇.

The ActRIIa variants of the invention preferably have reduced, weak, orno substantial binding to BMP9. BMP9 binding is reduced in ActRIIavariants containing the amino acid sequence TEEN at positions X₂₃, X₂₄,X₂₅, and X₂₆, as well as in variants that maintain the amino acid K atposition X₂₄ and have the amino acid sequence TKEN at positions X₂₃,X₂₄, X₂₅, and X₂₆. The sequences TEEN and TKEN can be employedinterchangeably in the ActRIIa variants (e.g., the variants in Tables 1and 2, e.g., SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) of the inventionto provide reduced BMP9 binding.

The ActRIIa variants of the invention may further include a C-terminalextension (e.g., additional amino acids at the C-terminus). TheC-terminal extension can add one to six additional amino acids at theC-terminus (e.g., 1, 2, 3, 4, 5, 6 or more additional amino acids) toany of the variants shown in Tables 1 and 2 (e.g., SEQ ID NOs: 1-70(e.g., SEQ ID NOs: 6-70)). One potential C-terminal extension that canbe included in the ActRIIa variants of the invention is amino acidsequence NP. For example, the sequence including the C-terminalextension is SEQ ID NO: 71 (e.g., SEQ ID NO: 69 with a C-terminalextension of NP). Another exemplary C-terminal extension that can beincluded in the ActRIIa variants of the invention is amino acid sequenceNPVTPK (SEQ ID NO: 155). For example, the sequence including theC-terminal extension is SEQ ID NO: 72 (e.g., SEQ ID NO: 69 with aC-terminal extension of NPVTPK (SEQ ID NO: 155)).

TABLE 1Amino acid substitutions in an extracellular ActRIIa variant havinga sequence of any one of SEQ ID NOs: 1-5GAILGRSETQECLX₁X₂NANWX₃X₄X₅X₆TNQTGVEX₇CX₈GX₉X₁₀X₁₁X₁₂X₁₃X₁₄HCX₁₅ATWX₁₆NISGSIEIVX₁₇X₁₈GCX₁₉X₂₀X₂₁DX₂₂NCYDRTDCVEX₂₃X₂₄X₂₅X₂₆PX₂₇VYFCCCEGNMCNEKFSYFPEMEVTQPTS(SEQ ID NO: 1)GAILGRSETQECLFX₂NANWX₃X₄X₅X₆TNQTGVEX₇CX₈GX₉KX₁₁X₁₂X₁₃X₁₄HCX₁₅ATWX₁₆NISGSIEIVX₁₇X₁₈GCX₁₉X₂₀X₂₁DX₂₂NCYDRTDCVEX₂₃X₂₄X₂₅X₂₆PX₂₇VYFCCCEGNMCNEKFSYFPEMEVTQPTS(SEQ ID NO: 2)GAILGRSETQECLFX₂NANWEX₄X₅RTNQTGVEX₇CX₈GX₉KDKRX₁₄HCX₁₅ATWX₁₆NISGSIEIVKX₁₈GCWLDDX₂₂NCYDRTDCVEX₂₃X₂₄X₂₅X₂₆PX₂₇VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 3)GAILGRSETQECLFX₂NANWEX₄DRTNQTGVEX₇CX₈GX₉KDKRX₁₄HCX₁₅ATWX₁₆NISGSIEIVKX₁₈GCWLDDX₂₂NCYDRTDCVEX₂₃KX₂₅X₂₆PX₂₇VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 4)GAILGRSETQECLFX₂NANWEX₄DRTNQTGVEPCX₈GX₉KDKRX₁₄HCFATWKNISGSIEIVKX₁₈GCWLDDINCYDRTDCVEX₂₃KX₂₅X₂₆PX₂₇VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 5) X₁F or Y X₁₅ F or Y x₂ F or Y X₁₆ K, R, or A x₃ E or A X₁₇K, A, Y, F, or I x₄ K or L X₁₈ Q or K X₅ D or E X₁₉ W or A X₆ R or A X₂₀L or A x₇ P or R X₂₁ D, K, R, A, F, G, M, N, or IGAILGRSETQECLX₁X₂NANWX₃X₄X₅X₆TNQTGVEX₇CX₈GX₉X₁₀X₁₁X₁₂X₁₃X₁₄HCX₁₅ATWX₁₆NISGSIEIVX₁₇X₁₈GCX₁₉X₂₀X₂₁DX₂₂NCYDRTDCVEX₂₃X₂₄X₂₅X₂₆PX₂₇VYFCCCEGNMCNEKFSYFPEMEVTQPTS(SEQ ID NO: 1)GAILGRSETQECLFX₂NANWX₃X₄X₅X₆TNQTGVEX₇CX₈GX₉KX₁₁X₁₂X₁₃X₁₄HCX₁₅ATWXINISGSIEIVX₁₇X₁₈GCX₁₉X₂₀X₂₁DX₂₂NCYDRTDCVEX₂₃X₂₄X₂₅X₂₆PX₂₇VYFCCCEGNMCNEKFSYFPEMEVTQPTS(SEQ ID NO: 2)GAILGRSETQECLFX₂NANWEX₄X₅RTNQTGVEX₇CX₈GX₉KDKRX₁₄HCX₁₅ATWX₁₆NISGSIEIVKX₁₈GCWLDDX₂₂NCYDRTDCVEX₂₃X₂₄X₂₅X₂₆PX₂₇VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 3)GAILGRSETQECLFX₂NANWEX₄DRTNQTGVEX₇CX₈GX₉KDKRX₁₄HCX₁₅ATWX₁₆NISGSIEIVKX₁₈GCWLDDX₂₂NCYDRTDCVEX₂₃KX₂₅X₂₆PX₂₇VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 4)GAILGRSETQECLFX₂NANWEX₄DRTNQTGVEPCX₈GX₉KDKRX₁₄HCFATWKNISGSIEIVKX₁₈GCWLDDINCYDRTDCVEX₂₃KX₂₅X₂₆PX₂₇VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 5) X₈Y or E X₂₂ I, F, or A X₉ D or E X₂₃ K or T X₁₀ K or Q X₂₄ K or E X₁₁D or A X₂₅ D or E X₁₂ K or A X₂₆ S or N X₁₃ R or A X₂₇ E or Q X₁₄ R or L

In some embodiments of the extracellular ActRIIa variant having thesequence of SEQ ID NO: 2, X₃ is E, X₆ is R, X₁₁ is D, X₁₂ is K, X₁₃ isR, X₁₆ is K or R, X₁₇ is K, X₁₉ is W, X₂₀ is L, X₂₁ is D, and X₂₂ is Ior F. In some embodiments of the extracellular ActRIIa variant havingthe sequence of SEQ ID NO: 1 or 2, X₁₇ is K. In some embodiments of theextracellular ActRIIa variant having the sequence of SEQ ID NOs: 1-3,X₁₇ is K, X₂₃ is T, X₂₄ is E, X₂₅ is E, and X₂₆ is N. In someembodiments of the extracellular ActRIIa variant having the sequence ofany one of SEQ ID NOs: 1-5, X₁₇ is K, X₂₃ is T, X₂₄ is K, X₂₅ is E, andX₂₆ is N.

In some embodiments, a polypeptide described herein includes anextracellular ActRIIa variant having a sequence of any one of SEQ IDNOs: 6-72 (Table 2).

TABLE 2Extracellular ActRIIa variants having the sequences of SEQ ID NOs: 6-72SEQ ID NO Amino Acid Sequence  6GAILGRSETQECLFYNANWELDRTNQTGVEPCEGEKDKRLHCFATWKNISGSIEIVKKGCWLDDINCYDRTDCVETKENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS  7GAILGRSETQECLFYNANWELERTNQTGVEPCEGEKDKRLHCFATWKNISGSIEIVKKGCWLDDINCYDRTDCVETKENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS  8GAILGRSETQECLFYNANWELDRTNQTGVERCEGEKDKRLHCFATWKNISGSIEIVKKGCWLDDINCYDRTDCVETKENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS  9GAILGRSETQECLFYNANWELDRTNQTGVEPCEGEKDKRLHCYATWKNISGSIEIVKKGCWLDDINCYDRTDCVETKENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 10GAILGRSETQECLFYNANWELDRTNQTGVEPCEGEKDKRLHCFATWRNISGSIEIVKKGCWLDDINCYDRTDCVETKENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 11GAILGRSETQECLFYNANWELDRTNQTGVEPCEGEKDKRLHCFATWKNISGSIEIVKKGCWLDDFNCYDRTDCVETKENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 12GAILGRSETQECLFYNANWELDRTNQTGVEPCEGEKDKRLHCFATWKNISGSIEIVKKGCWLDDINCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 13GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCFATWKNISGSIEIVKKGCWLDDINCYDRTDCVETKENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 14GAILGRSETQECLFYNANWELERTNQTGVEPCEGEKDKRLHCYATWKNISGSIEIVKKGCWLDDINCYDRTDCVETKENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 15GAILGRSETQECLFYNANWELERTNQTGVEPCEGEKDKRLHCFATWRNISGSIEIVKKGCWLDDINCYDRTDCVETKENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 16GAILGRSETQECLFYNANWELERTNQTGVEPCEGEKDKRLHCFATWKNISGSIEIVKKGCWLDDFNCYDRTDCVETKENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 17GAILGRSETQECLFYNANWELERTNQTGVEPCEGEKDKRLHCFATWKNISGSIEIVKKGCWLDDINCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 18GAILGRSETQECLFYNANWELDRTNQTGVERCEGEKDKRLHCYATWKNISGSIEIVKKGCWLDDINCYDRTDCVETKENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 19GAILGRSETQECLFYNANWELDRTNQTGVERCEGEKDKRLHCFATWRNISGSIEIVKKGCWLDDINCYDRTDCVETKENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 20GAILGRSETQECLFYNANWELDRTNQTGVERCEGEKDKRLHCFATWKNISGSIEIVKKGCWLDDFNCYDRTDCVETKENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 21GAILGRSETQECLFYNANWELDRTNQTGVERCEGEKDKRLHCFATWKNISGSIEIVKKGCWLDDINCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 22GAILGRSETQECLFYNANWELDRTNQTGVEPCEGEKDKRLHCYATWRNISGSIEIVKKGCWLDDINCYDRTDCVETKENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 23GAILGRSETQECLFYNANWELDRTNQTGVEPCEGEKDKRLHCYATWKNISGSIEIVKKGCWLDDFNCYDRTDCVETKENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 24GAILGRSETQECLFYNANWELDRTNQTGVEPCEGEKDKRLHCYATWKNISGSIEIVKKGCWLDDINCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 25GAILGRSETQECLFYNANWELDRTNQTGVEPCEGEKDKRLHCFATWRNISGSIEIVKKGCWLDDFNCYDRTDCVETKENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 26GAILGRSETQECLFYNANWELDRTNQTGVEPCEGEKDKRLHCFATWRNISGSIEIVKKGCWLDDINCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 27GAILGRSETQECLFYNANWELDRTNQTGVEPCEGEKDKRLHCFATWKNISGSIEIVKKGCWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 28GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCYATWKNISGSIEIVKKGCWLDDINCYDRTDCVETKENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 29GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCFATWRNISGSIEIVKKGCWLDDINCYDRTDCVETKENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 30GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCFATWKNISGSIEIVKKGCWLDDFNCYDRTDCVETKENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 31GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCFATWKNISGSIEIVKKGCWLDDINCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 32GAILGRSETQECLFYNANWELERTNQTGVEPCEGEKDKRLHCYATWRNISGSIEIVKKGCWLDDINCYDRTDCVETKENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 33GAILGRSETQECLFYNANWELERTNQTGVEPCEGEKDKRLHCYATWKNISGSIEIVKKGCWLDDFNCYDRTDCVETKENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 34GAILGRSETQECLFYNANWELERTNQTGVEPCEGEKDKRLHCYATWKNISGSIEIVKKGCWLDDINCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 35GAILGRSETQECLFYNANWELERTNQTGVEPCEGEKDKRLHCFATWRNISGSIEIVKKGCWLDDFNCYDRTDCVETKENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 36GAILGRSETQECLFYNANWELERTNQTGVEPCEGEKDKRLHCFATWRNISGSIEIVKKGCWLDDINCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 37GAILGRSETQECLFYNANWELERTNQTGVEPCEGEKDKRLHCFATWKNISGSIEIVKKGCWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 38GAILGRSETQECLFYNANWELDRTNQTGVERCEGEKDKRLHCYATWRNISGSIEIVKKGCWLDDINCYDRTDCVETKENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 39GAILGRSETQECLFYNANWELDRTNQTGVERCEGEKDKRLHCYATWKNISGSIEIVKKGCWLDDFNCYDRTDCVETKENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 40GAILGRSETQECLFYNANWELDRTNQTGVERCEGEKDKRLHCYATWKNISGSIEIVKKGCWLDDINCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 41GAILGRSETQECLFYNANWELDRTNQTGVERCEGEKDKRLHCFATWRNISGSIEIVKKGCWLDDFNCYDRTDCVETKENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 42GAILGRSETQECLFYNANWELDRTNQTGVERCEGEKDKRLHCFATWRNISGSIEIVKKGCWLDDINCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 43GAILGRSETQECLFYNANWELDRTNQTGVERCEGEKDKRLHCFATWKNISGSIEIV KKGCWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 44GAILGRSETQECLFYNANWELDRTNQTGVEPCEGEKDKRLHCYATWRNISGSIEIVKKGCWLDDFNCYDRTDCVETKENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 45GAILGRSETQECLFYNANWELDRTNQTGVEPCEGEKDKRLHCYATWRNISGSIEIVKKGCWLDDINCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 46GAILGRSETQECLFYNANWELDRTNQTGVEPCEGEKDKRLHCYATWKNISGSIEIVKKGCWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 47GAILGRSETQECLFYNANWELDRTNQTGVEPCEGEKDKRLHCFATWRNISGSIEIVKKGCWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 48GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCYATWRNISGSIEIVKKGCWLDDINCYDRTDCVETKENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 49GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCYATWKNISGSIEIVKKGCWLDDFNCYDRTDCVETKENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 50GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCYATWKNISGSIEIVKKGCWLDDINCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 51GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCFATWRNISGSIEIVKKGCWLDDFNCYDRTDCVETKENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 52GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCFATWRNISGSIEIVKKGCWLDDINCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 53GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCFATWKNISGSIEIVKKGCWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 54GAILGRSETQECLFYNANWELERTNQTGVEPCEGEKDKRLHCYATWRNISGSIEIVKKGCWLDDFNCYDRTDCVETKENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 55GAILGRSETQECLFYNANWELERTNQTGVEPCEGEKDKRLHCYATWRNISGSIEIVKKGCWLDDINCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 56GAILGRSETQECLFYNANWELERTNQTGVEPCEGEKDKRLHCYATWKNISGSIEIVKKGCWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 57GAILGRSETQECLFYNANWELERTNQTGVEPCEGEKDKRLHCFATWRNISGSIEIVKKGCWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 58GAILGRSETQECLFYNANWELDRTNQTGVERCEGEKDKRLHCYATWRNISGSIEIVKKGCWLDDFNCYDRTDCVETKENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 59GAILGRSETQECLFYNANWELDRTNQTGVERCEGEKDKRLHCYATWRNISGSIEIVKKGCWLDDINCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 60GAILGRSETQECLFYNANWELDRTNQTGVERCEGEKDKRLHCYATWKNISGSIEIVKKGCWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 61GAILGRSETQECLFYNANWELDRTNQTGVERCEGEKDKRLHCFATWRNISGSIEIVKKGCWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 62GAILGRSETQECLFYNANWELDRTNQTGVEPCEGEKDKRLHCYATWRNISGSIEIVKKGCWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 63GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCYATWRNISGSIEIVKKGCWLDDFNCYDRTDCVETKENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 64GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCYATWRNISGSIEIVKKGCWLDDINCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 65GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCYATWKNISGSIEIVKKGCWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 66GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCFATWRNISGSIEIVKKGCWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 67GAILGRSETQECLFYNANWELDRTNQTGVERCEGEKDKRLHCYATWRNISGSIEIVKKGCWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 68GAILGRSETQECLFYNANWELERTNQTGVEPCEGEKDKRLHCYATWRNISGSIEIVKKGCWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 69GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCYATWRNISGSIEIVKKGCWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 70GAILGRSETQECLYYNANWELERTNQTGVERCEGEQDKRLHCYATWRNISGSIEIVKKGCWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS 71GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCYATWRNISGSIEIVKKGCWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTSN P 72GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCYATWRNISGSIEIVKKGCWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTSN PVTPK

In some embodiments, a polypeptide of the invention including anextracellular ActRIIa variant (e.g., any one of SEQ ID NOs: 1-72 (e.g.,SEQ ID NOs: 6-72)) has amino acid K at position X₁₇. Altering the aminoacid at position X₁₇ can result in reduced activity. For example, anActRIIa variant having the sequenceGAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCYATWRNISGSIEIVAKGCWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 150) has reducedactivity in vivo, indicating that the substitution of A for K at X₁₇ isnot tolerated.

In some embodiments, a polypeptide of the invention including anextracellular ActRIIa variant (e.g., any one of SEQ ID NOs: 1-72 (e.g.,SEQ ID NOs: 6-72)) with the sequence TEEN at positions X₂₃, X₂₄, X₂₅,and X₂₆ can have a substitution of the amino acid K for the amino acid Eat position X₂₄. In some embodiments, a polypeptide of the inventionincluding an extracellular ActRIIa variant (e.g., any one of SEQ ID NOs:1-72 (e.g., SEQ ID NOs: 6-72)) with the sequence TKEN at positions X₂₃,X₂₄, X₂₅, and X₂₆ can have a substitution of the amino acid E for theamino acid K at position X₂₄. Polypeptides having the sequence TEEN orTKEN at positions X₂₃, X₂₄, X₂₅, and X₂₆ have reduced or weak binding toBMP9.

In some embodiments, a polypeptide of the invention including anextracellular ActRIIa variant (e.g., any one of SEQ ID NOs: 1-70 (e.g.,SEQ ID NOs: 6-70)) may further include a C-terminal extension (e.g.,additional amino acids at the C-terminus). In some embodiments, theC-terminal extension is amino acid sequence NP. For example, thesequence including the C-terminal extension is SEQ ID NO: 71 (e.g., SEQID NO: 69 with a C-terminal extension of NP). In some embodiments, theC-terminal extension is amino acid sequence NPVTPK (SEQ ID NO: 155). Forexample, the sequence including the C-terminal extension is SEQ ID NO:72 (e.g., SEQ ID NO: 69 with a C-terminal extension of NPVTPK (SEQ IDNO: 155)). The C-terminal extension can add one to six additional aminoacids at the C-terminus (e.g., 1, 2, 3, 4, 5, 6 or more additional aminoacids).

In some embodiments, a polypeptide of the invention including anextracellular ActRIIa variant may further include a moiety (e.g., Fcdomain monomer, a wild-type Fc domain, an Fc domain with amino acidsubstitutions (e.g., one or more substitutions that reducedimerization), an albumin-binding peptide, a fibronectin domain, or ahuman serum albumin), which may be fused to the N- or C-terminus (e.g.,C-terminus) of the extracellular ActRIIa variant by way of a linker orother covalent bonds. A polypeptide including an extracellular ActRIIavariant fused to an Fc domain monomer may form a dimer (e.g., homodimeror heterodimer) through the interaction between two Fc domain monomers,which combine to form an Fc domain in the dimer.

In some embodiments, an extracellular ActRIIa variant described hereindoes not have the sequence of any one of SEQ ID NOs: 76-96 shown inTable 3 below.

TABLE 3 Excluded Extracellular ActRIIa Variants. SEQ ID NOAmino Acid Sequence 76GAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWANISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTS 77GAILGRSETQECLFFNANWAKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTS 78GAILGRSETQECLFFNANWEKDATNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTS 79GAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKAKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTS 80GAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDARRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTS 81GAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKARHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTS 82GAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVAQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTS 83GAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVYQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTS 84GAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVFQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTS 85GAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVIQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTS 86GAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCALDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTS 87GAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWADDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTS 88GAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLKDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTS 89GAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLRDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTS 90GAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLADINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTS 91GAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLFDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTS 92GAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLGDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTS 93GAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLMDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTS 94GAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLNDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTS 95GAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLIDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTS 96GAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDANCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTS

Furthermore, in some embodiments, a polypeptide described herein has aserum half-life of at least 7 days in humans. The polypeptide may bindto bone morphogenetic protein 9 (BMP9) with a K_(D) of 200 pM or higher.The polypeptide may bind to activin A with a K_(D) of 10 pM or higher.In some embodiments, the polypeptide does not bind to BMP9 or activin A.In some embodiments, the polypeptide binds to activin and/or myostatinand exhibits reduced (e.g., weak) binding to BMP9. In some embodiments,the polypeptide that has reduced or weak binding to BMP9 has thesequence TEEN or TKEN at positions X₂₃, X₂₄, X₂₅, and X₂₆.

Additionally, in some embodiments, the polypeptide may bind to humanBMP9 with a K_(D) of about 200 pM or higher (e.g., a K_(D) of about 200,300, 400, 500, 600, 700, 800, or 900 pM or higher, e.g., a K_(D) ofabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 nM or higher,e.g., a K_(D) of between about 200 pM and about 50 nM). In someembodiments, the polypeptide does not substantially bind to human BMP9.In some embodiments, the polypeptide may bind to human activin A with aK_(D) of about 800 pM or less (e.g., a K_(D) of about 800, 700, 600,500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6,5, 4, 3, 2, or 1 pM or less, e.g., a K_(D) of between about 800 pM andabout 200 pM). In some embodiments, the polypeptide may bind to humanactivin B with a K_(D) of 800 pM or less (e.g., a K_(D) of about 800,700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10,9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less, e.g., a K_(D) of between about800 pM and about 200 pM) The polypeptide may also bind to growth anddifferentiation factor 11 (GDF-11) with a K_(D) of approximately 5 pM orhigher (e.g., a K_(D) of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130,135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200pM or higher).

II. Fc Domains

In some embodiments, a polypeptide described herein may include anextracellular ActRIIa variant fused to an Fc domain monomer of animmunoglobulin or a fragment of an Fc domain to increase the serumhalf-life of the polypeptide. A polypeptide including an extracellularActRIIa variant fused to an Fc domain monomer may form a dimer (e.g.,homodimer or heterodimer) through the interaction between two Fc domainmonomers, which form an Fc domain in the dimer. As conventionally knownin the art, an Fc domain is the protein structure that is found at theC-terminus of an immunoglobulin. An Fc domain includes two Fc domainmonomers that are dimerized by the interaction between the C_(H)3antibody constant domains. A wild-type Fc domain forms the minimumstructure that binds to an Fc receptor, e.g., FcγRI, FcγRIIa, FcγRIIb,FcγRIIIa, FcγRIIIb, FcγRIV. In some embodiments, an Fc domain may bemutated to lack effector functions, typical of a “dead” Fc domain. Forexample, an Fc domain may include specific amino acid substitutions thatare known to minimize the interaction between the Fc domain and an Fcγreceptor. In some embodiments, an Fc domain is from an IgG1 antibody andincludes amino acid substitutions L234A, L235A, and G237A. In someembodiments, an Fc domain is from an IgG1 antibody and includes aminoacid substitutions D265A, K322A, and N434A. The aforementioned aminoacid positions are defined according to Kabat (Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)). The Kabat numbering ofamino acid residues may be determined for a given antibody by alignmentat regions of homology of the sequence of the antibody with a “standard”Kabat numbered sequence. Furthermore, in some embodiments, an Fc domaindoes not induce any immune system-related response. For example, the Fcdomain in a dimer of a polypeptide including an extracellular ActRIIavariant fused to an Fc domain monomer may be modified to reduce theinteraction or binding between the Fc domain and an Fcγ receptor. Thesequence of an Fc domain monomer that may be fused to an extracellularActRIIa variant is shown below (SEQ ID NO: 97):

THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

In some embodiments, an Fc domain is from an IgG1 antibody and includesamino acid substitutions L12A, L13A, and G15A, relative to the sequenceof SEQ ID NO: 97. In some embodiments, an Fc domain is from an IgG1antibody and includes amino acid substitutions D43A, K100A, and N212A,relative to the sequence of SEQ ID NO: 97. In some embodiments, anextracellular ActRIIa variant described herein (e.g., an extracellularActRIIa variant having the sequence of any one of SEQ ID NOs: 1-72(e.g., SEQ ID NOs: 6-72)) may be fused to the N- or C-terminus of an Fcdomain monomer (e.g., SEQ ID NO: 97) through conventional genetic orchemical means, e.g., chemical conjugation. If desired, a linker (e.g.,a spacer) can be inserted between the extracellular ActRIIa variant andthe Fc domain monomer. The Fc domain monomer can be fused to the N- orC-terminus (e.g., C-terminus) of the extracellular ActRIIa variant.

In some embodiments, a polypeptide described herein may include anextracellular ActRIIa variant fused to an Fc domain. In someembodiments, the Fc domain contains one or more amino acid substitutionsthat reduce or inhibit Fc domain dimerization. In some embodiments, theFc domain contains a hinge domain. The Fc domain can be ofimmunoglobulin antibody isotype IgG, IgE, IgM, IgA, or IgD.Additionally, the Fc domain can be an IgG subtype (e.g., IgG1, IgG2a,IgG2b, IgG3, or IgG4). The Fc domain can also be a non-naturallyoccurring Fc domain, e.g., a recombinant Fc domain.

Methods of engineering Fc domains that have reduced dimerization areknown in the art. In some embodiments, one or more amino acids withlarge side-chains (e.g., tyrosine or tryptophan) may be introduced tothe C_(H)3-C_(H)3 dimer interface to hinder dimer formation due tosteric clash. In other embodiments, one or more amino acids with smallside-chains (e.g., alanine, valine, or threonine) may be introduced tothe C_(H)3-C_(H)3 dimer interface to remove favorable interactions.Methods of introducing amino acids with large or small side-chains inthe C_(H)3 domain are described in, e.g., Ying et al. (J Biol Chem.287:19399-19408, 2012), U.S. Patent Publication No. 2006/0074225, U.S.Pat. Nos. 8,216,805 and 5,731,168, Ridgway et al. (Protein Eng.9:617-612, 1996), Atwell et al. (J Mol Biol. 270:26-35, 1997), andMerchant et al. (Nat Biotechnol. 16:677-681, 1998), all of which areincorporated herein by reference in their entireties.

In yet other embodiments, one or more amino acid residues in the C_(H)3domain that make up the C_(H)3-C_(H)3 interface between two Fc domainsare replaced with positively-charged amino acid residues (e.g., lysine,arginine, or histidine) or negatively-charged amino acid residues (e.g.,aspartic acid or glutamic acid) such that the interaction becomeselectrostatically unfavorable depending on the specific charged aminoacids introduced. Methods of introducing charged amino acids in theC_(H)3 domain to disfavor or prevent dimer formation are described in,e.g., Ying et al. (J Biol Chem. 287:19399-19408, 2012), U.S. PatentPublication Nos. 2006/0074225, 2012/0244578, and 2014/0024111, all ofwhich are incorporated herein by reference in their entireties.

In some embodiments of the invention, an Fc domain includes one or moreof the following amino acid substitutions: T366W, T366Y, T394W, F405W,Y349T, Y349E, Y349V, L351T, L351H, L351N, L352K, P353S, S354D, D356K,D356R, D356S, E357K, E357R, E357Q, S364A, T366E, L368T, L368Y, L368E,K370E, K370D, K370Q, K392E, K392D, T394N, P395N, P396T, V397T, V397Q,L398T, D399K, D399R, D399N, F405T, F405H, F405R, Y407T, Y407H, Y407I,K409E, K409D, K409T, and K409I, relative to the sequence of human IgG1.In one particular embodiment, an Fc domain includes the amino acidsubstitution T366W, relative to the sequence of human IgG1. The sequenceof wild-type Fc domain is shown in SEQ ID NO: 151.

III. Albumin-Binding Peptide

In some embodiments, a polypeptide described herein may include anextracellular ActRIIa variant fused to a serum protein-binding peptide.Binding to serum protein peptides can improve the pharmacokinetics ofprotein pharmaceuticals.

As one example, albumin-binding peptides that can be used in the methodsand compositions described here are generally known in the art. In oneembodiment, the albumin binding peptide includes the sequenceDICLPRWGCLW (SEQ ID NO: 152).

In the present invention, albumin-binding peptides may be joined to theN- or C-terminus (e.g., C-terminus) of an extracellular ActRIIa variantdescribed herein (e.g., an extracellular ActRIIa variant having thesequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) toincrease the serum half-life of the extracellular ActRIIa variant. Insome embodiments, an albumin-binding peptide is joined, either directlyor through a linker, to the N- or C-terminus of an extracellular ActRIIavariant.

In some embodiments, an extracellular ActRIIa variant described herein(e.g., an extracellular ActRIIa variant having the sequence of any oneof SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) may be fused to the N- orC-terminus of albumin-binding peptide (e.g., SEQ ID NO: 152) throughconventional genetic or chemical means, e.g., chemical conjugation. Ifdesired, a linker (e.g., a spacer) can be inserted between theextracellular ActRIIa variant and the albumin-binding peptide. Withoutbeing bound to a theory, it is expected that inclusion of analbumin-binding peptide in an extracellular ActRIIa variant describedherein may lead to prolonged retention of the therapeutic proteinthrough its binding to serum albumin.

IV. Fibronectin Domain

In some embodiments, a polypeptide described herein may include anextracellular ActRIIa variant fused to fibronectin domains. Binding tofibronectin domains can improve the pharmacokinetics of proteinpharmaceuticals.

Fibronectin domain is a high molecular weight glycoprotein of theextracellular matrix, or a fragment thereof, that binds to, e.g.,membrane-spanning receptor proteins such as integrins and extracellularmatrix components such as collagens and fibrins. In some embodiments ofthe present invention, a fibronectin domain is joined to the N- orC-terminus (e.g., C-terminus) of an extracellular ActRIIa variantdescribed herein (e.g., an extracellular ActRIIa variant having thesequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) toincrease the serum half-life of the extracellular ActRIIa variant. Afibronectin domain can be joined, either directly or through a linker,to the N- or C-terminus of an extracellular ActRIIa variant.

As one example, fibronectin domains that can be used in the methods andcompositions described here are generally known in the art. In oneembodiment, the fibronectin domain is a fibronectin type III domain (SEQID NO: 153) having amino acids 610-702 of the sequence of UniProt ID NO:P02751. In another embodiment, the fibronectin domain is an adnectinprotein.

In some embodiments, an extracellular ActRIIa variant described herein(e.g., an extracellular ActRIIa variant having the sequence of any oneof SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) may be fused to the N- orC-terminus of a fibronectin domain (e.g., SEQ ID NO: 153) throughconventional genetic or chemical means, e.g., chemical conjugation. Ifdesired, a linker (e.g., a spacer) can be inserted between theextracellular ActRIIa variant and the fibronectin domain. Without beingbound to a theory, it is expected that inclusion of a fibronectin domainin an extracellular ActRIIa variant described herein may lead toprolonged retention of the therapeutic protein through its binding tointegrins and extracellular matrix components such as collagens andfibrins.

V. Serum Albumin

In some embodiments, a polypeptide described herein may include anextracellular ActRIIa variant fused to serum albumin. Binding to serumalbumins can improve the pharmacokinetics of protein pharmaceuticals.

Serum albumin is a globular protein that is the most abundant bloodprotein in mammals. Serum albumin is produced in the liver andconstitutes about half of the blood serum proteins. It is monomeric andsoluble in the blood. Some of the most crucial functions of serumalbumin include transporting hormones, fatty acids, and other proteinsin the body, buffering pH, and maintaining osmotic pressure needed forproper distribution of bodily fluids between blood vessels and bodytissues. In preferred embodiments, serum albumin is human serum albumin.In some embodiments of the present invention, a human serum albumin isjoined to the N- or C-terminus (e.g., C-terminus) of an extracellularActRIIa variant described herein (e.g., an extracellular ActRIIa varianthaving the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs:6-72)) to increase the serum half-life of the extracellular ActRIIavariant. A human serum albumin can be joined, either directly or througha linker, to the N- or C-terminus of an extracellular ActRIIa variant.

As one example, serum albumins that can be used in the methods andcompositions described herein are generally known in the art. In oneembodiment, the serum albumin includes the sequence of UniProt ID NO:P02768 (SEQ ID NO: 154).

In some embodiments, an extracellular ActRIIa variant described herein(e.g., an extracellular ActRIIa variant having the sequence of any oneof SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) may be fused to the N- orC-terminus of a human serum albumin (e.g., SEQ ID NO: 154) throughconventional genetic or chemical means, e.g., chemical conjugation. Ifdesired, a linker (e.g., a spacer) can be inserted between theextracellular ActRIIa variant and the human serum albumin. Without beingbound to a theory, it is expected that inclusion of a human serumalbumin in an extracellular ActRIIa variant described herein may lead toprolonged retention of the therapeutic protein.

VI. Linkers

A polypeptide described herein may include an extracellular ActRIIavariant (e.g., an extracellular ActRIIa variant having a sequence of anyone of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) fused to a moiety byway of a linker. In some embodiments, the moiety increases stability ofthe polypeptide. Exemplary moieties include an Fc domain monomer, awild-type Fc domain, an Fc domain with amino acid substitutions (e.g.,one or more substitutions that reduce dimerization), an albumin-bindingpeptide, a fibronectin domain, or a human serum albumin. In the presentinvention, a linker between a moiety (e.g., an Fc domain monomer (e.g.,the sequence of SEQ ID NO: 97), a wild-type Fc domain (e.g., SEQ ID NO:151), an Fc domain with amino acid substitutions (e.g., one or moresubstitutions that reduce dimerization), an albumin-binding peptide(e.g., SEQ ID NO: 152), a fibronectin domain (e.g., SEQ ID NO: 153), ora human serum albumin (e.g., SEQ ID NO: 154)) and an extracellularActRIIa variant (e.g., an extracellular ActRIIa variant having thesequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)), canbe an amino acid spacer including 1-200 amino acids. Suitable peptidespacers are known in the art, and include, for example, peptide linkerscontaining flexible amino acid residues such as glycine, alanine, andserine. In some embodiments, a spacer can contain motifs, e.g., multipleor repeating motifs, of GA, GS, GG, GGA, GGS, GGG, GGGA (SEQ ID NO: 98),GGGS (SEQ ID NO: 99), GGGG (SEQ ID NO: 100), GGGGA (SEQ ID NO: 101),GGGGS (SEQ ID NO: 102), GGGGG (SEQ ID NO: 103), GGAG (SEQ ID NO: 104),GGSG (SEQ ID NO: 105), AGGG (SEQ ID NO: 106), or SGGG (SEQ ID NO: 107).In some embodiments, a spacer can contain 2 to 12 amino acids includingmotifs of GA or GS, e.g., GA, GS, GAGA (SEQ ID NO: 108), GSGS (SEQ IDNO: 109), GAGAGA (SEQ ID NO: 110), GSGSGS (SEQ ID NO: 111), GAGAGAGA(SEQ ID NO: 112), GSGSGSGS (SEQ ID NO: 113), GAGAGAGAGA (SEQ ID NO:114), GSGSGSGSGS (SEQ ID NO: 115), GAGAGAGAGAGA (SEQ ID NO: 116), andGSGSGSGSGSGS (SEQ ID NO: 117). In some embodiments, a spacer can contain3 to 12 amino acids including motifs of GGA or GGS, e.g., GGA, GGS,GGAGGA (SEQ ID NO: 118), GGSGGS (SEQ ID NO: 119), GGAGGAGGA (SEQ ID NO:120), GGSGGSGGS (SEQ ID NO: 121), GGAGGAGGAGGA (SEQ ID NO: 122), andGGSGGSGGSGGS (SEQ ID NO: 123). In yet some embodiments, a spacer cancontain 4 to 12 amino acids including motifs of GGAG (SEQ ID NO: 104),GGSG (SEQ ID NO: 105), e.g., GGAG (SEQ ID NO: 104), GGSG (SEQ ID NO:105), GGAGGGAG (SEQ ID NO: 124), GGSGGGSG (SEQ ID NO: 125), GGAGGGAGGGAG(SEQ ID NO: 126), and GGSGGGSGGGSG (SEQ ID NO: 127). In someembodiments, a spacer can contain motifs of GGGGA (SEQ ID NO: 101) orGGGGS (SEQ ID NO: 102), e.g., GGGGAGGGGAGGGGA (SEQ ID NO: 128) andGGGGSGGGGSGGGGS (SEQ ID NO: 129). In some embodiments of the invention,an amino acid spacer between a moiety (e.g., an Fc domain monomer, awild-type Fc domain, an Fc domain with amino acid substitutions (e.g.,one or more substitutions that reduce dimerization), an albumin-bindingpeptide, a fibronectin domain, or a human serum albumin) and anextracellular ActRIIa variant (e.g., an extracellular ActRIIa varianthaving the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs:6-72)) may be GGG, GGGA (SEQ ID NO: 98), GGGG (SEQ ID NO: 100), GGGAG(SEQ ID NO: 130), GGGAGG (SEQ ID NO: 131), or GGGAGGG (SEQ ID NO: 132).

In some embodiments, a spacer can also contain amino acids other thanglycine, alanine, and serine, e.g., AAAL (SEQ ID NO: 133), AAAK (SEQ IDNO: 134), AAAR (SEQ ID NO: 135), EGKSSGSGSESKST (SEQ ID NO: 136),GSAGSAAGSGEF (SEQ ID NO: 137), AEAAAKEAAAKA (SEQ ID NO: 138),KESGSVSSEQLAQFRSLD (SEQ ID NO: 139), GENLYFQSGG (SEQ ID NO: 140),SACYCELS (SEQ ID NO: 141), RSIAT (SEQ ID NO: 142), RPACKIPNDLKQKVMNH(SEQ ID NO: 143), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 144),AAANSSIDLISVPVDSR (SEQ ID NO: 145), orGGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 146). In someembodiments, a spacer can contain motifs, e.g., multiple or repeatingmotifs, of EAAAK (SEQ ID NO: 147). In some embodiments, a spacer cancontain motifs, e.g., multiple or repeating motifs, of proline-richsequences such as (XP)_(n), in which X may be any amino acid (e.g., A,K, or E) and n is from 1-5, and PAPAP (SEQ ID NO: 148).

The length of the peptide spacer and the amino acids used can beadjusted depending on the two protein involved and the degree offlexibility desired in the final protein fusion polypeptide. The lengthof the spacer can be adjusted to ensure proper protein folding and avoidaggregate formation.

VII. Vectors, Host Cells, and Protein Production

The polypeptides of the invention can be produced from a host cell. Ahost cell refers to a vehicle that includes the necessary cellularcomponents, e.g., organelles, needed to express the polypeptides andfusion polypeptides described herein from their corresponding nucleicacids. The nucleic acids may be included in nucleic acid vectors thatcan be introduced into the host cell by conventional techniques known inthe art (e.g., transformation, transfection, electroporation, calciumphosphate precipitation, direct microinjection, infection, or the like).The choice of nucleic acid vectors depends in part on the host cells tobe used. Generally, preferred host cells are of either eukaryotic (e.g.,mammalian) or prokaryotic (e.g., bacterial) origin.

Nucleic Acid Vector Construction and Host Cells

A nucleic acid sequence encoding the amino acid sequence of apolypeptide of the invention may be prepared by a variety of methodsknown in the art. These methods include, but are not limited to,oligonucleotide-mediated (or site-directed) mutagenesis and PCRmutagenesis. A nucleic acid molecule encoding a polypeptide of theinvention may be obtained using standard techniques, e.g., genesynthesis. Alternatively, a nucleic acid molecule encoding a wild-typeextracellular ActRIIa may be mutated to include specific amino acidsubstitutions using standard techniques in the art, e.g., QuikChange™mutagenesis. Nucleic acid molecules can be synthesized using anucleotide synthesizer or PCR techniques.

A nucleic acid sequence encoding a polypeptide of the invention may beinserted into a vector capable of replicating and expressing the nucleicacid molecule in prokaryotic or eukaryotic host cells. Many vectors areavailable in the art and can be used for the purpose of the invention.Each vector may include various components that may be adjusted andoptimized for compatibility with the particular host cell. For example,the vector components may include, but are not limited to, an origin ofreplication, a selection marker gene, a promoter, a ribosome bindingsite, a signal sequence, the nucleic acid sequence encoding protein ofinterest, and a transcription termination sequence.

In some embodiments, mammalian cells may be used as host cells for theinvention. Examples of mammalian cell types include, but are not limitedto, human embryonic kidney (HEK) (e.g., HEK293, HEK 293F), Chinesehamster ovary (CHO), HeLa, COS, PC3, Vero, MC3T3, NSO, Sp2/0, VERY, BHK,MDCK, W138, BT483, Hs578T, HTB2, BT20, T47D, NSO (a murine myeloma cellline that does not endogenously produce any immunoglobulin chains),CRL7030, and HsS78Bst cells. In some embodiments, E. coli cells may alsobe used as host cells for the invention. Examples of E. coli strainsinclude, but are not limited to, E. coli 294 (ATCC®31,446), E. coli A1776 (ATCC®31,537, E. coli BL21 (DE3) (ATCC® BAA-1025), and E. coliRV308 (ATCC®31,608). Different host cells have characteristic andspecific mechanisms for the posttranslational processing andmodification of protein products (e.g., glycosylation). Appropriate celllines or host systems may be chosen to ensure the correct modificationand processing of the polypeptide expressed. The above-describedexpression vectors may be introduced into appropriate host cells usingconventional techniques in the art, e.g., transformation, transfection,electroporation, calcium phosphate precipitation, and directmicroinjection. Once the vectors are introduced into host cells forprotein production, host cells are cultured in conventional nutrientmedia modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.Methods for expression of therapeutic proteins are known in the art,see, for example, Paulina Balbas, Argelia Lorence (eds.) RecombinantGene Expression: Reviews and Protocols (Methods in Molecular Biology),Humana Press; 2nd ed. 2004 and Vladimir Voynov and Justin A. Caravella(eds.) Therapeutic Proteins: Methods and Protocols (Methods in MolecularBiology) Humana Press; 2nd ed. 2012.

Protein Production, Recovery, and Purification

Host cells used to produce the polypeptides of the invention may begrown in media known in the art and suitable for culturing of theselected host cells. Examples of suitable media for mammalian host cellsinclude Minimal Essential Medium (MEM), Dulbecco's Modified Eagle'sMedium (DMEM), Expi293™ Expression Medium, DMEM with supplemented fetalbovine serum (FBS), and RPMI-1640. Examples of suitable media forbacterial host cells include Luria broth (LB) plus necessarysupplements, such as a selection agent, e.g., ampicillin. Host cells arecultured at suitable temperatures, such as from about 20° C. to about39° C., e.g., from 25° C. to about 37° C., preferably 37° C., and CO₂levels, such as 5 to 10%. The pH of the medium is generally from about6.8 to 7.4, e.g., 7.0, depending mainly on the host organism. If aninducible promoter is used in the expression vector of the invention,protein expression is induced under conditions suitable for theactivation of the promoter.

In some embodiments, depending on the expression vector and the hostcells used, the expressed protein may be secreted from the host cells(e.g., mammalian host cells) into the cell culture media. Proteinrecovery may involve filtering the cell culture media to remove celldebris. The proteins may be further purified. A polypeptide of theinvention may be purified by any method known in the art of proteinpurification, for example, by chromatography (e.g., ion exchange,affinity, and size-exclusion column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. For example, the protein can be isolated andpurified by appropriately selecting and combining affinity columns suchas Protein A column (e.g., POROS Protein A chromatography) withchromatography columns (e.g., POROS HS-50 cation exchangechromatography), filtration, ultra filtration, salting-out and dialysisprocedures.

In other embodiments, host cells may be disrupted, e.g., by osmoticshock, sonication, or lysis, to recover the expressed protein. Once thecells are disrupted, cell debris may be removed by centrifugation orfiltration. In some instances, a polypeptide can be conjugated to markersequences, such as a peptide to facilitate purification. An example of amarker amino acid sequence is a hexa-histidine peptide (His-tag), whichbinds to nickel-functionalized agarose affinity column with micromolaraffinity. Other peptide tags useful for purification include, but arenot limited to, the hemagglutinin “HA” tag, which corresponds to anepitope derived from influenza hemagglutinin protein (Wilson et al.,Cell 37:767, 1984).

Alternatively, the polypeptides of the invention can be produced by thecells of a subject (e.g., a human), e.g., in the context of genetherapy, by administrating a vector (such as a viral vector (e.g., aretroviral vector, adenoviral vector, poxviral vector (e.g., vacciniaviral vector, such as Modified Vaccinia Ankara (MVA)), adeno-associatedviral vector, and alphaviral vector)) containing a nucleic acid moleculeencoding the polypeptide of the invention. The vector, once inside acell of the subject (e.g., by transformation, transfection,electroporation, calcium phosphate precipitation, direct microinjection,infection, etc.) will promote expression of the polypeptide, which isthen secreted from the cell. If treatment of a disease or disorder isthe desired outcome, no further action may be required. If collection ofthe protein is desired, blood may be collected from the subject and theprotein purified from the blood by methods known in the art.

VIII. Pharmaceutical Compositions and Preparations

The invention features pharmaceutical compositions that include thepolypeptides described herein (e.g., a polypeptide including anextracellular ActRIIa variant (e.g., an extracellular ActRIIa varianthaving the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs:6-72)). In some embodiments, a pharmaceutical composition of theinvention includes a polypeptide including an extracellular ActRIIavariant (e.g., an extracellular ActRIIa variant having the sequence ofany one of SEQ ID NOs: 1-70 (e.g., SEQ ID NOs: 6-70)) with a C-terminalextension (e.g., 1, 2, 3, 4, 5, 6 or more additional amino acids) as thetherapeutic protein. In some embodiments, a pharmaceutical compositionof the invention includes a polypeptide including an extracellularActRIIa variant (e.g., an extracellular ActRIIa variant having thesequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) fusedto a moiety (e.g., Fc domain monomer, or a dimer thereof, a wild-type Fcdomain, an Fc domain with amino acid substitutions (e.g., one or moresubstitutions that reduce dimerization), an albumin-binding peptide, afibronectin domain, or a human serum albumin) as the therapeuticprotein. In some embodiments, a pharmaceutical composition of theinvention including a polypeptide of the invention may be used incombination with other agents (e.g., therapeutic biologics and/or smallmolecules) or compositions in a therapy. In addition to atherapeutically effective amount of the polypeptide, the pharmaceuticalcomposition may include one or more pharmaceutically acceptable carriersor excipients, which can be formulated by methods known to those skilledin the art. In some embodiments, a pharmaceutical composition of theinvention includes a nucleic acid molecule (DNA or RNA, e.g., mRNA)encoding a polypeptide of the invention, or a vector containing such anucleic acid molecule.

Acceptable carriers and excipients in the pharmaceutical compositionsare nontoxic to recipients at the dosages and concentrations employed.Acceptable carriers and excipients may include buffers such asphosphate, citrate, HEPES, and TAE, antioxidants such as ascorbic acidand methionine, preservatives such as hexamethonium chloride,octadecyldimethylbenzyl ammonium chloride, resorcinol, and benzalkoniumchloride, proteins such as human serum albumin, gelatin, dextran, andimmunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone,amino acids such as glycine, glutamine, histidine, and lysine, andcarbohydrates such as glucose, mannose, sucrose, and sorbitol.Pharmaceutical compositions of the invention can be administeredparenterally in the form of an injectable formulation. Pharmaceuticalcompositions for injection can be formulated using a sterile solution orany pharmaceutically acceptable liquid as a vehicle. Pharmaceuticallyacceptable vehicles include, but are not limited to, sterile water,physiological saline, and cell culture media (e.g., Dulbecco's ModifiedEagle Medium (DMEM), α-Modified Eagles Medium (α-MEM), F-12 medium).Formulation methods are known in the art, see e.g., Banga (ed.)Therapeutic Peptides and Proteins: Formulation, Processing and DeliverySystems (3rd ed.) Taylor & Francis Group, CRC Press (2015).

The pharmaceutical compositions of the invention may be prepared inmicrocapsules, such as hydroxylmethylcellulose or gelatin-microcapsuleand poly-(methylmethacrylate) microcapsule. The pharmaceuticalcompositions of the invention may also be prepared in other drugdelivery systems such as liposomes, albumin microspheres,microemulsions, nano-particles, and nanocapsules. Such techniques aredescribed in Remington: The Science and Practice of Pharmacy 22^(th)edition (2012). The pharmaceutical compositions to be used for in vivoadministration must be sterile. This is readily accomplished byfiltration through sterile filtration membranes.

The pharmaceutical compositions of the invention may also be prepared asa sustained-release formulation. Suitable examples of sustained-releasepreparations include semipermeable matrices of solid hydrophobicpolymers containing the polypeptides of the invention. Examples ofsustained release matrices include polyesters, hydrogels, polyactides,copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as LUPRON DEPOT™, and poly-D-(−)-3-hydroxybutyric acid. Somesustained-release formulations enable release of molecules over a fewmonths, e.g., one to six months, while other formulations releasepharmaceutical compositions of the invention for shorter time periods,e.g., days to weeks.

The pharmaceutical composition may be formed in a unit dose form asneeded. The amount of active component, e.g., a polypeptide of theinvention, included in the pharmaceutical preparations is such that asuitable dose within the designated range is provided (e.g., a dosewithin the range of 0.01-100 mg/kg of body weight).

The pharmaceutical composition for gene therapy can be in an acceptablediluent, or can include a slow release matrix in which the gene deliveryvehicle is imbedded. If hydrodynamic injection is used as the deliverymethod, the pharmaceutical composition containing a nucleic acidmolecule encoding a polypeptide described herein or a vector (e.g., aviral vector) containing the nucleic acid molecule is delivered rapidlyin a large fluid volume intravenously. Vectors that may be used as invivo gene delivery vehicle include, but are not limited to, retroviralvectors, adenoviral vectors, poxviral vectors (e.g., vaccinia viralvectors, such as Modified Vaccinia Ankara), adeno-associated viralvectors, and alphaviral vectors.

IX. Routes, Dosage, and Administration

Pharmaceutical compositions that include the polypeptides of theinvention as the therapeutic proteins may be formulated for, e.g.,intravenous administration, parenteral administration, subcutaneousadministration, intramuscular administration, intra-arterialadministration, intrathecal administration, or intraperitonealadministration. The pharmaceutical composition may also be formulatedfor, or administered via, oral, nasal, spray, aerosol, rectal, orvaginal administration. For injectable formulations, various effectivepharmaceutical carriers are known in the art. See, e.g., ASHP Handbookon Injectable Drugs, Toissel, 18th ed. (2014).

In some embodiments, a pharmaceutical composition that includes anucleic acid molecule encoding a polypeptide of the invention or avector containing such nucleic acid molecule may be administered by wayof gene delivery. Methods of gene delivery are well-known to one ofskill in the art. Vectors that may be used for in vivo gene delivery andexpression include, but are not limited to, retroviral vectors,adenoviral vectors, poxviral vectors (e.g., vaccinia viral vectors, suchas Modified Vaccinia Ankara (MVA)), adeno-associated viral vectors, andalphaviral vectors. In some embodiments, mRNA molecules encodingpolypeptides of the invention may be administered directly to a subject.

In some embodiments of the present invention, nucleic acid moleculesencoding a polypeptide described herein or vectors containing suchnucleic acid molecules may be administered using a hydrodynamicinjection platform. In the hydrodynamic injection method, a nucleic acidmolecule encoding a polypeptide described herein is put under thecontrol of a strong promoter in an engineered plasmid (e.g., a viralplasmid). The plasmid is often delivered rapidly in a large fluid volumeintravenously. Hydrodynamic injection uses controlled hydrodynamicpressure in veins to enhance cell permeability such that the elevatedpressure from the rapid injection of the large fluid volume results influid and plasmid extravasation from the vein. The expression of thenucleic acid molecule is driven primarily by the liver. In mice,hydrodynamic injection is often performed by injection of the plasmidinto the tail vein. In certain embodiments, mRNA molecules encoding apolypeptide described herein may be administered using hydrodynamicinjection.

The dosage of the pharmaceutical compositions of the invention dependson factors including the route of administration, the disease to betreated, and physical characteristics, e.g., age, weight, generalhealth, of the subject. A pharmaceutical composition of the inventionmay include a dosage of a polypeptide of the invention ranging from 0.01to 500 mg/kg (e.g., 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450,or 500 mg/kg) and, in a more specific embodiment, about 0.1 to about 30mg/kg and, in a more specific embodiment, about 0.3 to about 30 mg/kg.The dosage may be adapted by the physician in accordance withconventional factors such as the extent of the disease and differentparameters of the subject.

The pharmaceutical compositions are administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective to result in an improvement or remediation of the symptoms.The pharmaceutical compositions are administered in a variety of dosageforms, e.g., intravenous dosage forms, subcutaneous dosage forms, andoral dosage forms (e.g., ingestible solutions, drug release capsules).Generally, therapeutic proteins are dosed at 0.1-100 mg/kg, e.g., 1-50mg/kg. Pharmaceutical compositions that include a polypeptide of theinvention may be administered to a subject in need thereof, for example,one or more times (e.g., 1-10 times or more) daily, weekly, biweekly,monthly, bimonthly, quarterly, biannually, annually, or as medicallynecessary. In some embodiments, pharmaceutical compositions that includea polypeptide of the invention may be administered to a subject in needthereof weekly, biweekly, monthly, bimonthly, or quarterly. Dosages maybe provided in either a single or multiple dosage regimens. The timingbetween administrations may decrease as the medical condition improvesor increase as the health of the patient declines.

X. Methods of Treatment

The invention is based on the discovery that substituting amino acidsfrom the extracellular portion of ActRIIb into the extracellular portionActRIIa yields ActRIIa variants with improved properties. The ActRIIavariants generated by introducing residues from ActRIIb into ActRIIaretain the beneficial properties of ActRIIa, such as longer serumhalf-life and low binding affinity to BMP9, and gain some of thebeneficial properties of ActRIIb, such as increased binding to activinsA and B (see Table 4) and an ability to increase muscle mass (seeExamples 1-3 and 5-6). These ActRIIa variant properties make for auseful therapeutic that can compete with endogenous activin receptorsfor ligand binding. As the ActRIIa variants contain the extracellularportion of the receptor, they will be soluble and able to bind to andsequester ligands (e.g., activins A and B, myostatin, GDF11) withoutactivating intracellular signaling pathways. Therefore, theextracellular ActRIIa variants can be used to treat diseases orconditions in which elevated activin signaling has been implicated(e.g., associated with increased expression of activin receptors oractivin receptor ligands). For example, loss of myostatin has been shownto increase skeletal muscle mass, suggesting that myostatin inhibitsskeletal muscle growth. It follows that treatment with a therapeuticagent that binds to myostatin and reduces its interaction withendogenous receptors could be a viable approach for increasing musclemass. Indeed, extracellular ActRIIa variants of the invention increasemuscle mass in mice (see Examples 1-3 and 5-6). These data indicate thatthe extracellular ActRIIa variants described herein can be used toincrease muscle mass and treat subjects with diseases or conditions thatresult in muscle weakness or atrophy.

Moreover, these data provide a compelling reason to use theextracellular ActRIIa variants of the invention to treat other diseasesor conditions associated with elevated expression of activin receptorsor activin receptor ligands, such as metabolic diseases (e.g., obesity,Type-1 diabetes, and Type-2 diabetes). Many studies have shown thatincreasing muscle mass is one way to reduce body fat and/or body weight,indicating that the extracellular ActRIIa variants described herein canbe used to treat metabolic diseases (e.g., obesity, Type-1 diabetes, andType-2 diabetes) indirectly by increasing muscle mass. However, asactivin receptors and activin receptor ligands have been shown to beincreased in in obese mice and humans, the extracellular ActRIIavariants described herein can be used to treat obesity by reducingelevated activin receptor signaling (e.g., by binding to andsequestering endogenous activin receptor ligands, e.g., activins andmyostatin).

The invention provides compositions and methods of treatment that may beused to increase muscle mass and strength in a subject in need thereof.In some embodiments, the subject may have a disease that results inmuscle weakness or atrophy (e.g., skeletal muscle weakness or atrophy).In some embodiments, the methods described herein are directed toaffecting myostatin, activin, and/or BMP9 signaling in a subject havinga disease or condition involving weakness and atrophy of muscles. Insome embodiments, a polypeptide including an extracellular ActRIIavariant described herein reduces or inhibits the binding of myostatin,activin, and/or BMP9 to their receptors, e.g., ActRIIa, ActRIIb, andBMPRII (e.g., ActRIIa). In some embodiments, affecting myostatin,activin, and/or BMP9 signaling (e.g., reducing or inhibiting the bindingof myostatin, activin, and/or BMP9 to their receptors, e.g., ActRIIa,ActRIIb, and BMPRII (e.g., ActRIIa)) results in an increase in thesubject's muscle mass.

In some embodiments, the polypeptides described herein (e.g., apolypeptide including an extracellular ActRIIa variant (e.g., anextracellular ActRIIa variant having the sequence of any one of SEQ IDNOs: 1-72 (e.g., SEQ ID NOs: 6-72)) may be administered to a subject toincrease muscle mass, or to affect myostatin, activin, and/or BMP9signaling in the subject. In some embodiments, the methods describedherein increase bone mineral density of the subject. In someembodiments, the methods described herein do not cause any vascularcomplications in the subject, such as increased vascular permeability orleakage. In some embodiments of the methods described herein, thesubject has a disease or condition involving weakness and atrophy ofmuscles (e.g., Duchenne muscular dystrophy (DMD), facioscapulohumeralmuscular dystrophy (FSHD), inclusion body myositis (IBM), amyotrophiclateral sclerosis (ALS), sarcopenia, or cancer cachexia).

The invention also includes methods of treating a subject havingDuchenne muscular dystrophy (DMD), facioscapulohumeral musculardystrophy (FSHD), inclusion body myositis (IBM), amyotrophic lateralsclerosis (ALS), sarcopenia, or cancer cachexia by administering to thesubject a polypeptide described herein (e.g., a polypeptide including anextracellular ActRIIa variant (e.g., an extracellular ActRIIa varianthaving the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs:6-72)).

The compositions and methods described herein can also be used to treatand/or prevent medical conditions, such as metabolic diseases, e.g.,obesity and diabetes (Type-1 and Type-2 diabetes). In some embodiments,the subject may have a disease that results in obesity. In someembodiments, the methods described herein are directed to affectingmyostatin, activin, and/or BMP9 signaling in a subject having obesity,diabetes (Type-1 and Type-2 diabetes), or a disease or condition thatresults in obesity. In some embodiments, a polypeptide including anextracellular ActRIIa variant described herein reduces or inhibits thebinding of myostatin, activin, and/or BMP9 to their receptors, e.g.,ActRIIa, ActRIIb, and BMPRII (e.g., ActRIIa). In some embodiments,affecting myostatin, activin, and/or BMP9 signaling (e.g., reducing orinhibiting the binding of myostatin, activin, and/or BMP9 to theirreceptors, e.g., ActRIIa, ActRIIb, and BMPRII (e.g., ActRIIa)) resultsin a reduction in the subject's body fat (e.g., amount of body fat orbody fat percentage), a reduction in the subject's body weight or bodyweight gain, a reduction in fasting insulin levels, an increase inglucose clearance, or an increase in insulin sensitivity (e.g., areduction in insulin resistance).

In some embodiments, the polypeptides described herein (e.g., apolypeptide including an extracellular ActRIIa variant (e.g., anextracellular ActRIIa variant having the sequence of any one of SEQ IDNOs: 1-72 (e.g., SEQ ID NOs: 6-72)) may be administered to a subject toprevent the development of obesity (e.g., in patients at risk ofdeveloping obesity, e.g., patients who are overweight, who have a familyhistory of obesity, or who have other medical conditions or genetic riskfactors linked to increased risk of obesity) and/or to treat patientsalready diagnosed with obesity. For example, administration of theextracellular ActRIIa variant (e.g., an extracellular ActRIIa varianthaving the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs:6-72)) to a subject may help to reduce the body weight of the subject bydecreasing the amount of fat. In some embodiments, the extracellularActRIIa variant decreases the amount of fat while maintaining orincreasing the amount of lean mass.

In some embodiments, the polypeptides described herein (e.g., apolypeptide including an extracellular ActRIIa variant (e.g., anextracellular ActRIIa variant having the sequence of any one of SEQ IDNOs: 1-72 (e.g., SEQ ID NOs: 6-72)) may be used to prevent thedevelopment of diabetes (e.g., Type-1 and Type-2 diabetes) and/or totreat patients already diagnosed with diabetes. Patients who are likelyto develop diabetes, e.g., individuals with genetic predisposition,family history of diabetes, prediabetes, association with otherautoimmune diseases, or other metabolic diseases, may be administeredthe polypeptides described herein (e.g., a polypeptide including anextracellular ActRIIa variant (e.g., an extracellular ActRIIa varianthaving the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs:6-72)) prophylactically, such that the extracellular ActRIIapolypeptides may maintain the normal function and health of p-cells andprevent or delay the autoimmune inflammatory damage to p-cells. In otherembodiments, the polypeptides described herein (e.g., a polypeptideincluding an extracellular ActRIIa variant (e.g., an extracellularActRIIa variant having the sequence of any one of SEQ ID NOs: 1-72(e.g., SEQ ID NOs: 6-72)) may be administered to individuals before theywould be diagnosed with diabetes (e.g., Type-1 and Type-2 diabetes) ordevelop clinical symptoms of diabetes, e.g., high blood glucose level,high fasting insulin level, insulin resistance, polyuria, polydipsia,and polyphagia. In some embodiments, the extracellular ActRIIapolypeptides may be administered to patients prior to the patientsneeding insulin. In yet other embodiments, the administration ofextracellular ActRIIa polypeptides may delay or postpone the need forinsulin treatment in diabetic patients. For example, administration ofthe extracellular ActRIIa polypeptides of the invention to a subject mayhelp to increase the rate of glucose clearance from the blood.

In some embodiments, the polypeptides described herein (e.g., apolypeptide including an extracellular ActRIIa variant (e.g., anextracellular ActRIIa variant having the sequence of any one of SEQ IDNOs: 1-72 (e.g., SEQ ID NOs: 6-72)) may be administered to a subject toprevent the development of and/or treat patients with obesity ordiabetes (e.g., Type-1 and Type-2 diabetes), or to affect myostatin,activin, and/or BMP9 signaling in the subject (e.g., to reduce orinhibit the binding of activin, myostatin, and/or BMP9 to theirreceptors). In some embodiments, the methods described herein reducebody fat (e.g., reduce the amount of subcutaneous and/or visceral fat,reduce adiposity, reduce the weights of epididymal and perirenal fatpads, or reduce body fat percentage). In some embodiments, the methodsdescribed herein reduce body weight or reduce body weight gain (e.g.,reduce the percentage of body weight gain). In some embodiments, themethods described herein reduce the proliferation of adipose cells. Insome embodiments, the methods described herein reduce LDL. In someembodiments, the methods described herein reduce triglycerides. In someembodiments, the methods described herein improve the serum lipidprofile of the subject. In some embodiments, the methods describedherein reduce body fat without reducing lean mass (e.g., do not affectlean mass or increase lean mass). In some embodiments, the methodsdescribed herein reduce body fat and increase muscle mass. In someembodiments, the methods described herein reduce blood glucose levels(e.g., fasting glucose levels) or and/or increase glucose clearance. Insome embodiments, the methods described herein reduce fasting insulinlevels and/or improve insulin sensitivity (e.g., reduce insulinresistance). In some embodiments, the methods described herein regulateinsulin biosynthesis and/or secretion from p-cells. In some embodiments,the methods described herein do not affect the appetite for food intake.In some embodiments, the methods described herein do not cause anyvascular complications in the subject, such as increased vascularpermeability or leakage.

In some embodiments, the polypeptides described herein (e.g., apolypeptide including an extracellular ActRIIa variant (e.g., anextracellular ActRIIa variant having the sequence of any one of SEQ IDNOs: 1-72 (e.g., SEQ ID NOs: 6-72)) decrease body fat, decrease bodyweight, or increase insulin sensitivity and/or glucose clearance byincreasing muscle mass.

In any of the methods described herein, a polypeptide including anextracellular ActRIIa variant (e.g., an extracellular ActRIIa varianthaving the sequence of any one of SEQ ID NOs: 1-71 (e.g., SEQ ID NOs:6-71)) that further includes a C-terminal extension of one to six aminoacids (e.g., 1, 2, 3, 4, 5, 6 or more amino acids) may be used as thetherapeutic protein. In any of the methods described herein, a dimer(e.g., homodimer or heterodimer) of a polypeptide including anextracellular ActRIIa variant (e.g., an extracellular ActRIIa varianthaving the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs:6-72)) fused to a moiety (e.g., an Fc domain monomer, a wild-type Fcdomain, an Fc domain with amino acid substitutions (e.g., one moresubstitutions that reduce dimerization), an albumin-binding peptide, afibronectin domain, or a human serum albumin) may be used as thetherapeutic protein. Nucleic acids encoding the polypeptides describedherein, or vectors containing said nucleic acids can also beadministered according to any of the methods described herein. In any ofthe methods described herein, the polypeptide, nucleic acid, or vectorcan be administered as part of a pharmaceutical composition.

EXAMPLES Example 1—Effect of Extracellular ActRIIa Variants on BodyWeight

C57Bl/6 mice received a single hydrodynamic injection of a plasmidconstruct encoding one of the following six polypeptides (n=10/group):

-   -   (1) human Fc (hFc),    -   (2) extracellular ActRIIa (SEQ ID NO: 73) fused to the        N-terminus of hFc through a GGG linker;    -   (3) extracellular ActRIIb (SEQ ID NO: 74) fused to the        N-terminus of hFc through a GGG linker;    -   (4) extracellular ActRIIa variant (SEQ ID NO: 69) fused to the        N-terminus of hFc through a GGG linker; and    -   (5) extracellular ActRIIb variant (SEQ ID NO: 149) fused to the        N-terminus of hFc through a GGG linker.

100 μg of plasmid construct was delivered in a volume of 10% body weightover 5-8 seconds. The high volume and short period of injection providesthe pressure needed to introduce the plasmid into the liver cells wherethe plasmid will be expressed, specifically the proteins of interest areexpressed under a strong and ubiquitous promoter. The protein ofinterest is secreted under the endogenous machinery of the liver cellsand circulates freely. Mice were weighted twice weekly for 30 days andmeasurements were recorded as absolute body weight (BW) in grams and asa percent of body weight change from baseline measurements (FIGS. 2A and2B, respectively).

Example 2—Effect of Extracellular ActRIIa Variants on Muscle Mass

Mice received a single hydrodynamic injection of a plasmid constructencoding one of the following six polypeptides (n=10/group):

-   -   (1) human Fc (hFc),    -   (2) extracellular ActRIIa (SEQ ID NO: 73) fused to the        N-terminus of hFc through a GGG linker;    -   (3) extracellular ActRIIb (SEQ ID NO: 74) fused to the        N-terminus of hFc through a GGG linker;    -   (4) extracellular ActRIIa variant (SEQ ID NO: 69) fused to the        N-terminus of hFc through a GGG linker; and    -   (5) extracellular ActRIIb variant (SEQ ID NO: 149) fused to the        N-terminus of hFc through a GGG linker.

100 μg of plasmid construct was delivered in a volume of 10% body weightover 5-8 seconds. The high volume and short period of injection providesthe pressure needed to introduce the plasmid into the liver cells wherethe plasmid will be expressed, specifically the proteins of interest areexpressed under a strong and ubiquitous promoter. The protein ofinterest is secreted under the endogenous machinery of the liver cellsand circulates freely. On study days 0 (baseline), 14, and 28, miceunderwent NMR analysis for determination of lean mass using a MiniSpecLF90 NMR analyzer (Bruker, Woodlands, Tex.). The percent lean masschanges from baseline were recorded on days 14 and 28 (FIGS. 3A and 3B).

Example 3—Effect of Extracellular ActRIIa Variants on Body Weight whenAdministered as Purified Recombinant Protein

Female C57Bl/6 mice (Taconic Biosciences, Hudson N.Y.) received anintraperitoneal injection of tris-buffered saline vehicle or one of thefollowing five purified recombinant polypeptides at a dosage of 10 mg/kgtwice weekly for four weeks (n=10/group):

-   -   (1) tris-buffered saline vehicle,    -   (2) extracellular ActRIIa (SEQ ID NO: 73) fused to the        N-terminus of hFc through a GGG linker;    -   (3) extracellular ActRIIb (SEQ ID NO: 74) fused to the        N-terminus of hFc through a GGG linker;    -   (4) extracellular ActRIIa/b variant (SEQ ID NO: 69) fused to the        N-terminus of hFc through a GGG linker;    -   (5) extracellular ActRIIa/bΔ9 variant (SEQ ID NO: 58) fused to        the N-terminus of hFc through a GGG linker; and    -   (6) extracellular ActRII a/bΔ9 min variant (SEQ ID NO: 6) fused        to the N-terminus of hFc through a GGG linker.

Purified recombinant protein was made by transient expression in HEK293cell and purified from the conditioned media using Protein-A Sepharosechromatography.

Following four weeks of dosing the mice were humanely sacrificed andnecropsy was performed. Necropsy included collection of weights fortotal body, and the gastrocnemius, pectoralis, and quadriceps muscles.Statistical analysis of muscle/body weight data was performed inGraphPad Prism 7 (GraphPad Software, La Jolla Calif.) (FIGS. 4A and 4B,respectively).

Example 4—Evaluation of ActRIIa Variants Binding Affinity by SurfacePlasmon Resonance (SPR)

The Biacore 3000 was used to measure the kinetics of the interactionsbetween the ActRIIa variants and the ligands Activin A, Activin B,growth differentiation factor 11 (GDF11), and BMP-9. ActRIIa variantswere expressed and purified according to the methodology described inExample 3. The ActRIIa variants were immobilized on the chip (CM4 orCM5) with capture antibodies (anti-mouse from GEGE) in flow cells 2-4 toensure proper orientation. Flow cell 1 was used as a reference cell tosubtract any nonspecific binding and bulk effects. HBS-EP+ buffer fromGE Healthcare™ was used as a running buffer. Each ligand was run in aconcentration series at 40 μl/min to avoid mass transport effects. Thedata was analyzed using Scrubber2 by BioLogic™ Software to calculate theK_(D) of each interaction (Table 4).

TABLE 4 Comparison of ActRIIa variant binding affinity (K_(D)) tovarious ligands Activin A (K_(D)) Activin B (K_(D)) GDF-11 (K_(D)) BMP-9(K_(D)) Vehicle N/A N/A N/A N/A ActRIIa 1 nM 373 pM 81 pM 25 nM (SEQ IDNO: 73) ActRIIb 63 pM 23 pM 115 pM 278 pM (SEQ ID NO: 74) ActRIIa/bvariant 542 pM 103 pM 186 pM 4 nM (SEQ ID NO: 69) ActRIIb/a variant NoBinding No Binding No Binding No Binding (SEQ ID NO: 149) ActRIIa/bΔ9variant 213 pM 12.3 pM 115 pM 10 nM (SEQ ID NO: 58) ActRIIa/bΔ9 min 310pM 88 pM 114 pM 17 nM variant (SEQ ID NO: 6) ActRIIa/b+ variant 242 pM282 pM No dissociation 26 nM (SEQ ID NO: 150) ActRIIa/bΔ9m2 variant 170pM 104 pM 222 pM 13-18 nM (SEQ ID NO: 38) ActRIIa/bΔ9m3 variant 71 pM72.5 pM 117 pM 1.2 nM (SEQ ID NO: 41) ActRIIa/bΔ9m4 variant 375 pM 254pM 394 pM 14-20 nM (SEQ ID NO: 44) ActRIIa/bmax1 variant 232 pM 97 pM236 pM 5.6 nM (SEQ ID NO: 70) ActRIIa/bmax2 variant 135 pM 39 pM 113 pM5 nM (SEQ ID NO: 71) ActRIIa/bmax3 variant 89 pM 43 pM 214 pM 3.3 nM(SEQ ID NO: 72) *Not done in HDI, but recombinant protein demonstratesthe BW result is similar to ActRIIa/b

Example 5—Effect of Extracellular ActRIIa Variants on Body and MuscleWeight

C57Bl/6 mice received a single hydrodynamic injection of a plasmidconstruct encoding one of the following twelve polypeptides(n=10/group):

-   -   (1) vehicle;    -   (2) pLEV113-ActRIIa (19-127) (SEQ ID NO: 73) fused to the        N-terminus of hFc through a GGG linker;    -   (3) pLEV113-ActRIIb (41-155) (SEQ ID NO: 74) fused to the        N-terminus of hFc through a GGG linker;    -   (4) pLEV113-ActRIIa/b (SEQ ID NO: 69) fused to the N-terminus of        hFc through a GGG linker;    -   (5) pLEV113-ActRIIb/a (SEQ ID NO: 149) fused to the N-terminus        of hFc through a GGG linker;    -   (6) pLEV113-ActRIIa/b+ (SEQ ID NO: 150) fused to the N-terminus        of hFc through a GGG linker;    -   (7) pLEV113-ActRIIa/b-delta 9m2 (SEQ ID NO: 38) fused to the        N-terminus of hFc through a GGG linker;    -   (8) pLEV113-ActRIIa/b-delta 9m3 (SEQ ID NO: 41) fused to the        N-terminus of hFc through a GGG linker;    -   (9) pLEV113-ActRIIa/b-delta 9m4 (SEQ ID NO: 44) fused to the        N-terminus of hFc through a GGG linker;    -   (10) pLEV113-ActRIIa/bmax1 (SEQ ID NO: 70) fused to the        N-terminus of hFc through a GGG linker;    -   (11) pLEV113-ActRIIa/bmax2 (SEQ ID NO: 71) fused to the        N-terminus of hFc through a GGG linker; and    -   (12) pLEV113-ActRIIa/bmax1 (SEQ ID NO: 72) fused to the        N-terminus of hFc through a GGG linker.

100 μg of plasmid construct was delivered in a volume of 10% body weightover 5-8 seconds. The high volume and short period of injection providesthe pressure needed to introduce the plasmid into the liver cells wherethe plasmid will be expressed, specifically the proteins of interest areexpressed under a strong and ubiquitous promoter. The protein ofinterest is secreted under the endogenous machinery of the liver cellsand circulates freely. Mice were weighted twice weekly for 30 days andmeasurements were recorded as absolute body weight (BW) in grams and asa percent of body weight change from baseline measurements (FIGS. 5A and5B, respectively). Muscles were also weighed at the end of the study andmeasurements were recorded in grams (FIGS. 6A and 6B).

Example 6—Dose Effect of Extracellular ActRIIa Variants on Body Weight,Muscle Weight, and Muscle Mass

8-week old, male C57BL/6 mice were weight-matched into 9 groups(n=10/group). Groups were dosed with 5 mL/kg of either vehicle(Tris-Buffered Saline, pH 7.4) or one of 4 concentrations ofActRIIA/B-Fc (SEQ ID NO: 69 fused to the N-terminus of hFc through a GGGlinker) or ActRIIA/BA9-Fc (SEQ ID NO: 58 fused to the N-terminus of hFcthrough a GGG linker). The doses evaluated were 20 mg/kg, 8 mg/kg, 3mg/kg and 1 mg/kg. Treatments were administered intraperitoneally (IP)twice a week for 4 weeks (8 doses), and the study was terminated onstudy day 28. Body weights were recorded on dosing days throughout thestudy (FIGS. 7A and 7B), and at study termination, groups underwent NMRimaging for lean and fat mass analysis (FIGS. 8A and 8B) and hadpectoralis and gastrocnemius muscles weights collected and weighed(FIGS. 9A and 9B).

Example 7—Effect of Extracellular ActRIIa Variants on Obesity

Adult male C57BL/6 mice are assigned to weight-matched treatment groups(n=10/group). All animals are maintained on either regular chow diet(Chow; Purina LabDiet 5001; St. Louis, Mo.) or high fat diet (HFD;Research Diets D12331; New Brunswick, N.J.). Chow- and HFD-fed groupsare further divided into groups that are dosed twice weekly with eitherActRII variant or vehicle for a period of 60 d. Body weights aremeasured twice per week at the time of treatment. Body composition ismeasured using the MiniSpec LF50 at baseline (before administration oftreatments and transfer to HFD) and then every other week until the endof the study. At the study termination date, tissues of interest (serum,plasma, muscles and fat depots) are surgically removed and weighed.Serum samples are subsequently evaluated for biomarkers of adiposity andplasma was evaluated for Hba1c levels.

OTHER EMBODIMENTS

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure come within known or customary practice within theart to which the invention pertains and may be applied to the essentialfeatures hereinbefore set forth.

All publications, patents, and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety. Other embodiments are within the following claims.

The invention claimed is:
 1. A nucleic acid molecule encoding apolypeptide comprising an extracellular activin receptor type IIa(ActRIIa) variant and an Fc domain, the variant having a sequence of(SEQ ID NO: 1) GAILGRSETQECLX₁X₂NANWX₃X₄X₅X₆TNQTGVEX₇CX₈GX₉X₁₀X₁₁X₁₂X₁₃X₁₄HCX₁₅ATWX₁₆NISGSIEIVX₁₇X₁₈GCX₁₉X₂₀X₂₁DX₂₂NCYDRTDCVEX₂₃X₂₄X₂₅X₂₆PX₂₇VYFCCCEGNMCNEKFSYFPEMEVTQPTS,

wherein X₁ is F or Y; X₂ is Y; X₃ is E; X₄ is L; X₅ is D or E; X₆ is R;X₇ is P or R; X₈ is E; X₉ is E; X₁₀ is K or Q; X₁₁ is D; X₁₂ is K; X₁₃is R; X₁₄ is L; X₁₅ is F or Y; X₁₆ is K or R; X₁₇ is K; X₁₈ is K; X₁₉ isW; X₂₀ is L; X₂₁ is D; X₂₂ is I or F; X₂₃ is T; X₂₄ is K or E; X₂₅ is E;X₂₆ is N; and X₂₇ is Q.
 2. The nucleic acid molecule of claim 1, whereinX₁ is F and X₁₀ is K.
 3. The nucleic acid molecule of claim 1, whereinthe variant has the sequence of any one of SEQ ID NOs: 6-72.
 4. Thenucleic acid molecule of claim 1, wherein the polypeptide furthercomprises a C-terminal extension of one or more amino acids at theC-terminus of the ActRIIa variant.
 5. The nucleic acid molecule of claim4, wherein the C-terminal extension is NP or NPVTPK (SEQ ID NO: 155). 6.The nucleic acid molecule of claim 1, wherein the Fc domain is fused tothe C-terminus of the ActRIIa variant by way of a linker.
 7. The nucleicacid molecule of claim 1, wherein the variant has the sequence of anyone of SEQ ID NOs: 6, 38, 41, 44, 58, 69, 70, 71, and
 72. 8. The nucleicacid molecule of claim 1, wherein the variant has the sequence of SEQ IDNO:
 69. 9. The nucleic acid molecule of claim 8, wherein the Fc domainis fused to the C-terminus of the ActRIIa variant by way of a linker.10. A vector comprising the nucleic acid molecule of claim
 1. 11. Thevector of claim 10, wherein the vector is a viral vector.
 12. The vectorof claim 11, wherein the viral vector is a retroviral vector, anadenoviral vector, a poxviral vector, an adeno-associated viral vector,or an alphaviral vector.